Human monoclonal antibodies specific for hepatitis C virus (HCV) E2 antigen

ABSTRACT

The present invention relates to compositions derived from immunoglobulin molecules specific for the hepatitis C virus (HCV). More particularly, the invention is related to molecules which are capable of specifically binding with HCV E2 antigen. The molecules are useful in specific binding assays, affinity purification schemes and pharmaceutical compositions for the prevention and treatment of HCV infection in mammalian subjects. The invention thus relates to novel human monoclonal antibodies specific for HCV E2 antigen, fragments of such monoclonal antibodies, polypeptides having structure and function substantially homologous to antigen-binding sites obtained from such monoclonal antibodies, nucleic acid molecules encoding those polypeptides, and expression vectors comprising the nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/635,109, filed Apr. 19, 1996, now U.S. Pat. No. 6,538,114 from whichpriority is claimed pursuant to 35 U.S.C. §120 and which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to compositions derived fromimmunoglobulin molecules specific for the hepatitis C virus (HCV). Moreparticularly, the invention is related to recombinant human monoclonalantibodies which are capable of specifically binding with HCV E2antigen.

BACKGROUND

Hepatitis C virus (HCV) infection occurs throughout the world and is themajor cause of transfusion-associated hepatitis. There are an estimated150,000 new cases of HCV infection each year in the United States. Theseroprevalence of anti-HCV antibodies in blood donors from around theworld has been shown to vary between 0.02 and 1.23%, with rates in somecountries as high as about 19%. In addition to being the predominatecause of transfusion-induced hepatitis, HCV is also a common cause ofhepatitis in individuals exposed to blood or blood products. Thus,recipients of blood or blood products, intravenous drug users, renaldialysis patients and needle-stick victims represent high-risk groupsfor HCV infection. Alter et al. (1993) Infect Agents Dis 2:155-166.Further, heterosexual transmission of HCV across the urogenital tract,and mother-to-baby transmission, has been well documented. Ohto et al.(1994) N Engl J Med 330:744-750. Other risk factors associated with HCVinfection include familial or household contact with an HCV-infectedindividual and health-care employment with occupational exposure toblood and hemodialysis. Alter et al. (1990) JAMA 264:2231-2235. Chronichepatitis develops in approximately 62% of infections. Alter et al.(1992) N Engl J Med 327:1899-1905.

Most of the serious liver disease associated with HCV results from thehigh propensity of the agent to cause chronic, persistent infection.Cirrhosis occurs in approximately 20% of chronic cases, of which 20 to25% will result in liver failure. Another serious sequela associatedwith HCV infection is primary hepatocellular carcinoma.

The viral genomic sequence of HCV is known, as are methods for obtainingthe sequence. See, e.g., International Publication Nos. WO 89/04669; WO90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense,single-stranded RNA genome and is a member of the Flaviridae family ofviruses. Currently, there are 6 distinct, but related genotypes of HCVwhich have been identified based on phylogenetic analyses (Simmonds etal., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a singlepolypeptide having more than 3000 amino acid residues (Choo et al.(1989) Science 244:359-362; Choo et al. (1991) Proc. Natl. Acad. Sci.USA 88:2451-2455; Han et al. (1991) Proc. Natl. Acad. Sci. USA88:1711-1715). The polypeptide is processed co- and post-translationallyinto both structural and non-structural (NS) proteins.

In particular, there are three putative HCV structural proteins,consisting of the N-terminal nucleocapsid protein (termed “core”) andtwo envelope glycoproteins, “E1” (also known as E) and “E2” (also knownas E2/NS1). (See, Houghton et al. (1991) Hepatology 14:381-388, for adiscussion of HCV proteins, including E1 and E2.) E1 is detected as a32-35 kDa species and is converted into a single endo H-sensitive bandof approximately 18 Kda. By contrast, E2 displays a complex pattern uponimmunoprecipitation consistent with the generation of multiple species(Grakoui et al. (1993) J. Virol. 67:1385-1395; Tomei et al. (1993) J.Virol. 67:4017-4026). The HCV envelope glycoproteins E1 and E2 form astable complex that is coimmunoprecipitable (Grakoui et al. (1993) J.Virol. 67:1385-1395; Lanford et al. (1993) Virology 197:225-235; Ralstonet al. (1993) J. Virol. 67:6753-6761).

The only currently available treatment for chronic hepatitis C infectionconsists of α-interferon (α-IFN) therapy. However, long-term response tointerferon therapy only occurs in 10% to 30% of treated individuals, andthere is evidence that the different HCV strains vary greatly in theirresponsiveness to interferon therapy, with the type 1 viruses being themost refractive. Furthermore, flu-like side effects are commonlyencountered with interferon therapy (occurring in approximately 60% to80% of treated individuals), as well as other less common side effectssuch as nausea, depression, fatigue and thrombocytopenia. Interferontherapy is also not indicated for immunocompromised individuals.Accordingly, there exists a need for more effective therapeuticapproaches in the treatment of chronic HCV infection. In this regard,some effect has been seen using ribivirin, or combination therapies withursodiol and α-IFN.

In particular, the HCV E1 and E2 proteins are of considerable interestbecause recombinant vaccines based on those molecules have been shown tobe protective against experimental challenge with HCV in primatestudies. (Choo et al. (1994) Proc. Natl. Acad. Sci. USA 91:1294-1298).Hyperimmune globulin compositions of anti-HCV antibody moleculesobtained from donor samples have been described for the treatment of HCVin infected individuals, and in the prevention of HCV infection inhigh-risk groups. European Patent Application Publication No. 447,984,published Sep. 25, 1991. Since these compositions are made from donorblood products, an inherent risk is associated with their use due to thepossible presence of infectious against such as the HumanImmunodeficiency Virus (HIV) and HCV. Accordingly, hyperimmune globulinpreparations must be carefully screened, and all infectious agentsinactivated prior to administration to human subjects.

It is known that the immune response to HCV in normal individualsincludes both humoral and cell mediated components. Koziel et al. (1993)J Virol 67:7522-7532, Alter et al. (1989) N Engl J Med 321:1494-1500.Further, several reports have indicated that antibodies elicited to HCVmay neutralize the infectivity of the virus. Shimizu et al. (1994) JVirol 68:1494-1500, Farci et al. (1994) Proc Natl Acad Sci USA91:7792-7796. Such results provide hope that an effective antibody-basedtherapy can be developed. In this regard, the administration of ahighly-reactive, neutralizing anti-HCV antibody preparation to anindividual who is at risk of infection, or who has been recently exposedto the agent will provide immediate passive immunity to the individual.Such passive immunizations would likewise be expected to be successfulin both normal and immunocompromised subjects. Preferably, theneutralizing antibodies would be broadly cross-reactive againstdifferent HCV strains, and would be monoclonal in order to control theeffects of the use of the antibodies in vivo.

For a number of practical and economic reasons, murine monoclonalantibodies have been generally used in research and medicine. Murineantibodies can be raised against a wide variety of molecules, such asHCV antigens, and fused with a myeloma cell to yield hybridomas whichcan be grown in culture to produce monoclonal antibodies toward HCVantigens. Kohler et al. (1975) Nature 256:495-497. Although suchmonoclonal antibodies may have antigen binding specificities ofsignificant therapeutic value, the use of such murine antibodies in thetreatment of human disease has been limited since those molecules areimmunogenic to the human immune system. Thus, murine monoclonals havebeen most commonly used in immunodiagnostics. In this regard, murinemonoclonal antibodies to putative HCV E2 envelope polypeptides have beendescribed for use in the detection of HCV in biological samples. U.S.Pat. No. 5,308,750 to Mehta et al.

Accordingly, there remains a need in the art to provide human monoclonalantibodies toward HCV E2 antigen, wherein the monoclonals are broadlycross-reactive with heterologous HCV isolates.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of human monoclonalantibody molecules which exhibit immunological binding affinity for HCVE2 polypeptide antigen, and which are cross-reactive against differentHCV strains. The monoclonal antibody molecules were obtained from acombinatorial library that was constructed from a nonimmunizedHCV-infected source. The present molecules generally comprise a humanantibody Fab molecule that exhibits immunological binding affinity forHCV E2 antigen.

Accordingly, in one embodiment, the invention is directed to arecombinant human monoclonal antibody that exhibits immunologicalbinding affinity for HCV E2 antigen, wherein the monoclonal antibodyincludes amino acid sequences that are homologous to the binding portionof a human antibody Fab molecule obtained from a combinatorial antibodylibrary. The recombinant monoclonal antibody molecule can be in the formof a substantially whole immunoglobulin molecule, or can be in the formof a soluble Fab molecule, an Fv fragment, or an sFv molecule, whereineach molecule at least contains amino acid sequences that are homologousto the binding portion of a human antibody Fab molecule.

In another embodiment, the invention is directed to an isolated nucleicacid molecule which contains a polynucleotide coding sequence for apolypeptide that is homologous to the binding portion of a heavy orlight chain variable region (V_(H) or V_(L)) of a human Fab moleculewhich exhibits immunological binding affinity for HCV E2 antigen. In arelated embodiment, the invention is directed to an isolated nucleicacid molecule which contains polynucleotide coding sequences for a firstpolypeptide and polynucleotide coding sequences for a secondpolypeptide, wherein the first polypeptide is homologous to the bindingportion of a heavy chain variable region (V_(H)) of a human Fab moleculewhich exhibits immunological binding affinity for HCV E2 antigen, andthe second polypeptide is homologous to the binding portion of a lightchain variable region (V_(L)) of a human Fab molecule which exhibitsimmunological binding affinity for the HCV E2 antigen.

In other embodiments, the invention pertains to expression vectorscomprising the nucleic acid molecules above operably linked to controlsequences that direct the transcription of the polynucleotide codingsequences when the vector is present in a host cell or under suitableconditions for the transcription and translation of the polynucleotidecoding sequences. Yet further embodiments of the invention pertain tohost cells transformed with the vectors of the invention, and methodsfor producing recombinant polypeptides using the transformed host cells.

In another embodiment, the invention is directed to vaccine compositionscomprising the recombinant monoclonal antibody molecules of theinvention. Still further embodiments relate to methods of using thevaccine compositions, wherein the vaccines are used to provide anantibody titer to HCV in a mammalian subject, and/or used to providepassive immunity against HCV infection in a vaccinated subject. Inrelated embodiments, the vaccine compositions are used in combinationwith known anti-HCV therapeutics.

In still further embodiments, the recombinant monoclonal antibodymolecules of the invention are used to provide binding complexes whichare labeled with a detectable moiety. The labeled binding complexes areused in related embodiments of the invention, such as in specificbinding assay methods, for detecting the presence of HCV particles insamples suspected of containing HCV and in specific binding assays formonitoring the progress of anti-HCV treatment of HCV-infected subjects.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1G depict the deduced γ1 heavy chain amino acid sequences ofthe Fab molecule clones 1:5 (SEQ ID NO:1), 1:7 (SEQ ID NO:2), 1:11 (SEQID NO:3), L3 (SEQ ID NO:4), L1 (SEQ ID NO:5), A8 (SEQ ID NO:6), and A12(SEQ ID NO:7), respectively. The CDR regions of each heavy chain havebeen identified in the Figures as “CDR1,” “CDR2” and “CDR3.”

FIGS. 2A-2G depict the deduced κ light chain amino acid sequences of theFab molecule clones 1:5 (SEQ ID NO:8), 1:7 (SEQ ID NO:9), 1:11 (SEQ IDNO:10), L3 (SEQ ID NO:11), L1 (SEQ ID NO:12), A8 (SEQ ID NO:13), and A12(SEQ ID NO:14), respectively. The CDR regions (CDR1, CDR2 and CDR3) ofeach light chain have also been identified as noted in regard to FIGS.1A-1G above.

FIGS. 3A-3G depict the κ light chain nucleic acid sequences of the Fabmolecule clones 1:5 (SEQ ID NO:15), 1:7 (SEQ ID NO:16), 1:11 (SEQ IDNO:17), L3 (SEQ ID NO:18), L1 (SEQ ID NO:19), A8 (SEQ ID NO:20), and A12(SEQ ID NO:21), respectively.

FIGS. 4A-4G depict the γ1 heavy chain nucleic acid sequences of the Fabmolecule clones 1:5 (SEQ ID NO:22), 1:7 (SEQ ID NO:23), 1:11 (SEQ IDNO:24), L3 (SEQ ID NO:25) L1 (SEQ ID NO:19), A8 (SEQ ID NO:26), and A12(SEQ ID NO:27), respectively.

FIG. 5 depicts the results of a neutralization assay conducted asdescribed in Example 12 wherein the ability of Fab molecules expressedfrom the Fab molecule clones 1:5, 1:7, 1:11 and L3 to block the bindingof HCV E2 polypeptide to target cells was assessed.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields andD. M. Knipe, eds.)

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, the terms “hepatitis C virus,” or “HCV” describe thevirus in a generic manner, and as such the terms are not limiting to anyparticular HCV viral sequence or isolate. In this regard, there are 6distinct genotypes of HCV with 11 distinct subtypes which have beenidentified based on phylogenetic analyses (Houghton, M. (1996)“Hepatitis C Viruses,” Fields Virology, 3d Edition, Fields et al. eds.,Lippincott-Raven Publishers, Philadelphia Pa.; Simmonds et al., J. Gen.Virol. (1993) 74:2391-2399). Further, comparison of genomic nucleotidesequences from different HCV isolates around the world establish thatHCV is highly heterogenous, with a range of sequence diversity among 74isolates. Thus, the terms “hepatitis C virus,” and “HCV” as used hereinwill generically encompass all such isolates.

The terms “an antigen derived from an E1 polypeptide,” an “E1polypeptide antigen” and “an HCV E1 antigen” are used interchangeablyherein and encompass molecules from an HCV E1 region. The term“polypeptide,” as used herein, refers to a polymer of amino acids anddoes not refer to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not refer to or exclude post expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. E1 polypeptides antigenscan be physically derived from the HCV E1 region or producedrecombinantly or synthetically, based on the known sequence. The matureE1 region of HCV1 begins at approximately amino acid 192 of thepolyprotein and continues to approximately amino acid 383.

A polypeptide or amino acid sequence “derived from” a designated HCVregion refers to a polypeptide having an amino acid sequence identicalto that of a polypeptide encoded in the sequence, or a portion thereofwherein the portion consists of at least 3-5 amino acids, preferably atleast 4-7 amino acids, more preferably at least 8-10 amino acids, andeven more preferably at least 11-15 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.This terminology also includes a polypeptide expressed from a designatedHCV region.

The terms “an antigen derived from an E2 polypeptide,” an “E2polypeptide antigen” and “an HCV E2 antigen” are used interchangeablyherein and encompass molecules from an HCV E2 region. Such molecules canbe physically derived from the region or produced recombinantly orsynthetically, based on the known sequence. The mature E2 region of HCV1is believed to begin at approximately amino acid 384-385.

For purposes of the present invention, HCV E1 and E2 polypeptides aredefined with respect to the amino acid number of the polyprotein encodedby the genome of HCV1, with the initiator methionine being designatedposition 1. However, it should be noted that an antigen from an “E1polypeptide” or an “E2 polypeptide” is not limited to polypeptideshaving an exact HCV1 sequence. Indeed, the HCV genome is in a state ofconstant flux and contains several variable domains which exhibitrelatively high degrees of variability between isolates. It is readilyapparent that the terms encompass antigens from E1 and E2 polypeptidesfrom any of the various HCV isolates including isolates having any ofthe 6 genotypes of HCV described in Simmonds et al., J Gen Virol (1993)74:2391-2399). In this regard, the corresponding E1 or E2 regions in aheterologous HCV isolate can be readily determined by aligning sequencesfrom the two isolates in a manner that brings the sequences into maximumalignment. This can be performed with any of a number of computersoftware packages, such as ALIGN 1.0, available from the University ofVirginia, Department of Biochemistry (Attn: Dr. William R. Pearson).See, Pearson et al., Proc Natl Acad Sci USA (1988) 85:2444-2448.

Additionally, the terms “E1 polypeptide antigen” and “E2 polypeptideantigen” encompass polypeptides which include modifications to thenative sequence, such as internal deletions, additions and substitutions(generally conservative in nature). These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through naturally occurring mutational events.

An “E1/E2 complex” refers to a complex of the E1 and E2 polypeptidesdescribed above. The mode of association of E1 and E2 in such a complexis immaterial. Indeed, such a complex may form spontaneously simply bymixing E1 and E2 polypeptides which have been produced individually.Similarly, when co-expressed and secreted, E1 and E2 polypeptides canform a complex spontaneously in the media. Formation of an “E1/E2complex” is readily determined using standard protein detectiontechniques such as polyacrylamide gel electrophoresis and immunologicaltechniques such as immunoprecipitation.

The term “antibody” encompasses monoclonal antibody preparations, aswell as preparations including hybrid antibodies, altered antibodies,F(ab′)₂ fragments, F(ab) molecules, Fv fragments, single domainantibodies, chimeric antibodies and functional fragments thereof whichexhibit immunological binding properties of the parent antibodymolecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited by the manner in which it is made. The term encompasses wholeimmunoglobulin molecules, as well as Fab molecules, F(ab′)₂ fragments,Fv fragments, and other molecules that exhibit immunological bindingproperties of the parent monoclonal antibody molecule. The term“recombinant monoclonal antibody” is defined herein as a monoclonalantibody that has been produced by expression of a recombinantpolynucleotide.

The term “antigen-binding site,” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev Biochem59:439-473.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. One suchmolecule is a Fab molecule which comprises a heterodimer that includesan intact antigen-binding site. The enzyme pepsin is able to cleave IgGmolecules to provide several fragments, including the “F(ab′)₂” fragmentwhich comprises both antigen-binding sites. An “Fv” fragment can beproduced by preferential proteolytic cleavage of an IgM, and on rareoccasions IgG or IgA immunoglobulin molecule. Fv fragments are, however,more commonly derived using recombinant techniques known in the art. TheFv fragment includes a non-covalent V_(H)::V_(L) heterodimer includingan antigen-binding site which retains much of the antigen recognitionand binding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A polypeptide molecule, or amino acid sequence “derived from” adesignated Fab molecule or Fab nucleic acid sequence refers to apolypeptide having an amino acid sequence identical to that of a Fabpolypeptide encoded in the sequence, or a portion thereof wherein theportion consists of at least 3-5 amino acids, preferably at least 4-7amino acids, more preferably at least 8-10 amino acids, and even morepreferably at least 11-15 amino acids.

A single chain Fv (“sFv”) polypeptide molecule is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883. A number ofmethods have been described to discern chemical structures forconverting the naturally aggregated, but chemically separated, light andheavy polypeptide chains from an antibody V region into an Sfv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRs and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, Frs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs.

By “purified” and “isolated” is meant, when referring to a polypeptideor nucleotide sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules of the same type.The terms “purified” and “isolated” as used herein preferably mean atleast 75% by weight, more preferably at least 85% by weight, morepreferably still at least 95% by weight, and most preferably at least98% by weight, of biological macromolecules of the same type arepresent. An “isolated nucleic acid molecule which encodes a particularpolypeptide” refers to a nucleic acid molecule which is substantiallyfree of other nucleic acid molecules that do not encode the subjectpolypeptide; however, the molecule may include some additional bases ormoieties which do not deleteriously affect the basic characteristics ofthe composition. Thus, for example, an isolated nucleic acid moleculewhich encodes the binding portion of a particular heavy chain variableregion of an antibody consists essentially of the nucleotide codingsequence for the subject binding portion (e.g., the CDR set interposedbetween the FR set).

“Homology” refers to the percent of identity between two polynucleotideor polypeptide moieties. The correspondence between two or moresequences can be determined by techniques known in the art. For example,homology can be determined by a direct comparison of the sequenceinformation between two polypeptide molecules. Alternatively, homologycan be determined by hybridization of polynucleotides under conditionswhich form stable duplexes between homologous regions (for example,those which would be used prior to S₁ digestion), followed by digestionwith single-stranded specific nuclease(s), followed by sizedetermination of the digested fragments. Two DNA or polypeptidesequences are “substantially homologous” when at least about 60%(preferably at least about 80%, and most preferably at least about 90%)of the nucleotides or amino acids match over a defined length of themolecule.

The terms “recombinant DNA molecule,” or “recombinant nucleic acidmolecule” are used herein to refer to a polynucleotide of genomic, cDNA,semisynthetic, or synthetic origin which, by virtue of its origin ormanipulation: (1) is not associated with all or a portion of apolynucleotide with which it is associated in nature, (2) is linked to apolynucleotide other than that to which it is linked in nature, or (3)does not occur in nature. Thus, the term encompasses “syntheticallyderived” nucleic acid molecules.

The term “nucleic acid molecule” as used herein refers to a polymericform of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule and thus includes double- and single-stranded DNA and RNA.It also includes known types of modifications, for example, labels whichare known in the art, methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including for e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.

A “coding sequence” is a nucleic acid molecule which is translated intoa polypeptide, usually via mRNA, when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequencemay be determined by a translation start codon at the 5′-terminus and atranslation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to, cDNA, and recombinant nucleotidesequences.

“Control sequence” refers to nucleic acid sequences which are necessaryto effect the expression of coding sequences to which they are ligated.The nature of such control sequences differs depending upon the hostorganism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is necessary for expression of a coding sequence, and may alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.

As used herein, the term “expression cassette” refers to a moleculecomprising at least one coding sequence operably linked to a controlsequence which includes all nucleotide sequences required for thetranscription of cloned copies of the coding sequence and thetranslation of the mRNAs in an appropriate host cell. Such expressioncassettes can be used to express eukaryotic genes in a variety of hostssuch as bacteria, blue-green algae, plant cells, yeast cells, insectcells and animal cells. Under the invention, expression cassettes caninclude, but are not limited to, cloning vectors, specifically designedplasmids, viruses or virus particles. The cassettes may further includean origin of replication for autonomous replication in host cells,selectable markers, various restriction sites, a potential for high copynumber and strong promoters.

By “vector” is meant any genetic element, such as a plasmid, phage,transposon, cosmid, chromosome, virus etc., which is capable ofreplication when associated with the proper control elements and whichcan transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

“Recombinant host cells”, “host cells,” “cells,” “cell cultures,” andother such terms denote, for example, microorganisms, insect cells, andmammalian cells, that can be, or have been, used as recipients forrecombinant vector or other transfer DNA, and include the progeny of theoriginal cell which has been transformed. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.Examples for mammalian host cells include Chinese hamster ovary (CHO)and monkey kidney (COS) cells.

Specifically, as used herein, “cell line,” refers to a population ofcells capable of continuous or prolonged growth and division in vitro.Often, cell lines are clonal populations derived from a singleprogenitor cell. It is further known in the art that spontaneous orinduced changes can occur in karyotype during storage or transfer ofsuch clonal populations. Therefore, cells derived from the cell linereferred to may not be precisely identical to the ancestral cells orcultures, and the cell line referred to includes such variants. The term“cell lines” also includes immortalized cells.

“Transformation”, as used herein, refers to the insertion of anexogenous polynucleotide into a host cell, irrespective of the methodused for the insertion, for example, direct uptake, transduction,f-mating or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host genome.

B. General Methods

The present invention is based on the generation of novel cross-genotypereactive human monoclonal antibody molecules specific to the HCV E2envelope glycoprotein. The monoclonal antibodies are obtained using acombinatorial antibody library constructed from a nonimmunized source,and are useful in the prevention, therapy and diagnosis of HCV infectionin mammalian subjects. More particularly, the monoclonal antibodies areobtained from combinatorial libraries expressing Fab molecules on thesurface of filamentous DNA bacteriophage using antigen selectiontechniques.

Preparation of Combinatorial Libraries

Combinatorial libraries for the purposes of the present invention can beconstructed using known techniques, such as those described by Chanocket al. (1993) Infect Agents Dis 2:118-131 and Barbas, III et al. (1995)Methods: Comp. Meth Enzymol 8:94-103. Antibody-producing cells can beobtained from an unimmunized, HCV-infected donor from, e.g., plasma,serum, spinal fluid, lymph fluid, the external sections of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells and myelomas. Preferably, the antibody-producing cellsource is lymphocytes that have been obtained from a bone marrow orperipheral blood sample of an unimmunized subject.

Lymphocytes can be obtained from the sample and total RNA isolated andextracted using known methods. See, e.g., Chomczynski et al. (1987) AnalBiochem 162:156-159. The RNA can be reverse-transcribed into firststrand cDNA using oligo-dT priming. The DNA encoding immunoglobulinheavy (Fd) and light chain fragments can be amplified using thepolymerase chain reaction (PCR) to provide all of the genetic materialnecessary to produce Fab antigen-binding molecules. Saiki, et al. (1986)Nature 324:163, Scharf et al. (1986) Science 233:1076-1078 and U.S. Pat.Nos. 4,683,195 and 4,683,202. In conducting the PCR amplification, anumber of known primers can preferably be used to select for γ1 heavychain and κ light chain sequences. Persson et al. (1991) Proc Natl AcadSci USA 88:2432-2436, Kang et al. (1991) Methods: Comp. Meth Enzymol2:111-118. The PCR products are pooled separately into heavy and lightchain DNA preparations, and then purified, for example, using gelelectrophoresis. The purified heavy and light chain DNA molecules arethen digested with suitable restriction enzymes, and the digestedproducts purified and ligated into a suitable phagemid vector system.Yang et al. (1995) J Mol Biol 254:392-403, Barbas, III et al. (1995)Methods: Comp. Meth Enzymol 8:94-103, Barbas, III et al. (1991) ProcNatl Acad Sci USA 88:7978-7982. A number of suitable phagemid vectorsystems are known in the art; however, a particularly preferred vectorfor use herein is the pComb3H vector which has been previouslydescribed. Barbas, III et al. (1995), supra. When the Pcomb3h phagemidvector is used, heavy chain DNA is cloned into the subject phagemidadjacent to, and upstream of, the sequence for the C-terminal anchoragedomain of the phagemid coat protein III (cpIII). The cpiii protein is anintegral membrane protein, and thus serves as a membrane anchor for theFab assembly.

The vectors generally include selectable markers known in the art. Forexample, the Pcomb3h phagemid vector contains the bacterial ampicillinresistance gene (B-lactamase). The vector will also include appropriatefirst and second leader sequences, respectively arranged upstream of theinsertion sites for the heavy and light chain coding sequences, wherebyexpression products from the heavy and light chain coding regions aretargeted to the periplasm when produced in a suitable host cell. InPcomb3h, these leader sequences are pelB sequences, omp A sequences orcombinations thereof.

The phagemid vector system containing the human immunoglobulin DNA isthen introduced into a suitable bacterial host cell (for example usingelectrophoresis), wherein the phagemid expresses a heavy chain-cpiiifusion polypeptide and a light chain polypeptide, each of which aretargeted to the periplasm of the host cell by their associated leadersequences. The transfected bacterial host cell containing the phagemidvector is selected by growth in a suitable medium containing a selectiveagent corresponding to the selectable marker of the phagemid vector(e.g., ampicillin).

Rescue of the phagemid DNA is conducted using known techniques. Inparticular, the transfected host cell is infected with a helper phagewhich encodes a number of expression products necessary in trans forpackaging the phagemid DNA into recombinant virus particles.Single-stranded copies of the phagemid DNA are thus packaged into viralparticles which, upon leaving the host cell, incorporate phage cpVIIImolecules and are capped by a limited number of phage cpiiimolecules—some of which cpviii and cpiii molecules are linked to Fabmolecules. Recombinant phage particles displaying Fab molecules (termed“phage-Fabs”) contain the corresponding heavy and light chain geneswithin the packaged genome.

When the above technique is practiced using an initial library ofphagemids, the rescue process generates a library of recombinant phagewhich display Fab molecules (a phage display library). The rescueprocess further results in amplification of the initial library, suchthat multiple copies of each recombinant phage clone (along with eachset of immunoglobulin heavy and light chain binding portions) aregenerated. The phage display library is then “panned” against HCV E2antigen to select for Fab molecules which are capable of selectivelybinding to that antigen. More particularly, the panning procedure can beconducted by applying a suspension containing the phage display libraryonto HCV E2 antigen that has been immobilized to a plastic reactionvessel according to known methods. Burton et al. (1991) Proc Natl AcadSci USA 88:10134-10137. After incubation under suitable bindingconditions, non-specifically bound phage particles are removed byrepeated washings. The resulting HCV E2-antigen specific phage-Fabs arethen eluted from the insoluble antigen using low Ph, or in the presenceof excess soluble E2 antigen. The panning procedure is repeated severaltimes, wherein bacterial host cells are infected by the eluted phageafter each round of panning to propagate phage-Fab clones for eachsubsequent round of panning. Samuelsson et al. (1995) Virology207:495-502.

In the present invention, the panning procedure was specificallydeveloped to select for highly potent, cross-genotype reactive Fabmolecules specific for HCV E2 antigen. In particular, the genotype ofserum HCV of the unimmunized, HCV-infected human subject from which theantibody-producing cells were obtained was determined using knownmethods. Widell et al. (1994) J Med Virol 44:272-279. Selection forstrain cross-reactivity was provided by experimental design, wherein thepanning procedure was conducted using HCV E2 antigen derived from adifferent HCV genotype than that of the HCV from the infected humandonor. Furthermore, the E2 antigen used in the panning procedures wasselected so as to provide HCV E2 antigen in substantially the sameconformation as expected for that antigen in vivo.

Two different recombinant HCV envelope protein preparations were used toprovide the selecting antigen in the above-described panning procedure,a “conformational” CHO E2 molecule, and a CHO E1/E2 complex. Theconformational E2 molecule was constructed, expressed and secreted fromrecombinant CHO cells as previously described in Spaete et al. (1992)Virology 188:819-830, then purified using known methods (Rosa et al.(1996) Proc Natl Acad Sci USA 93:1759-1763). A recombinant complexedE1/E2 preparation was constructed and expressed from recombinant CHOcells as described in Spaete et al. (1992) supra, then purified usingknown methods (Choo et al. (1994) Proc Natl Acad Sci USA 91:1294-1298).Once purified, the selecting antigens were immobilized to a plasticreaction vessel as described above.

Individual clones exhibiting superior binding affinity for the selectingantigen were selected, and expressed by growing infected host cells inthe selective medium until a suitable volume of cells was reached. Thebacterial host cells were pelleted and then resuspended in medium. Aftersuitable incubation, the cells were spun down, and the periplasmiccontent released by freeze-thawing techniques. After the bacterialdebris was removed by centrifugation, the Fab-containing supernatant wastransferred to suitable containers, and stored for future use.

Once the selected Fabs are expressed, binding characteristics of theselected Fab molecules can be determined. In particular, the affinity ofthe Fab molecules for HCV E2 antigen was determined herein using aninhibition ELISA technique. See, e.g., Persson et al. (1991) Proc NatlAcad Sci USA 88:2432-2436, Rath et al. (1988) J Immun Methods106:245-249. Clones that expressed Fab molecules of high potency (e.g.,an affinity of at least about 1×10⁷ M⁻¹, and preferably at least about1.7×10⁷ M⁻¹ as determined by inhibition ELISA) were identified forsequencing.

Phage (plasmid) DNA from clones which exhibited high potency binding inthe panning selection process was isolated, and single stranded DNA wasobtained by PCR using primers (one of which, e.g., is biotinylated atthe 5′ end) that hybridize upstream and downstream of the immunoglobulincloning regions. After PCR, single stranded DNA was obtained bydenaturing the DNA under alkaline conditions, and absorbing biotinylatedDNA strands onto a solid support. Dideoxy sequencing reactions wereperformed according to known methods (Sanger et al. (1977) Proc NatlAcad Sci USA 74:5463-5467) using labeled primers hybridizing 3′ of thejunction between the variable and constant regions. Kabat et al., inSequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. ofHealth and Human Services, U.S. Government Printing Office, 1987). Thereaction products were run on an automated sequencer (for example,A.L.F. available from Pharmacia Biotech). The nucleic acid sequenceinformation thus obtained was analyzed to provide coding sequences forthe heavy chain and light chain portions of the selected monoclonal Fabmolecules. Multiple copies of the same clones were identified bycomparisons of sequence data. Further, the deduced amino acid sequenceswere obtained using known methods.

Using the above nucleic acid sequence information, coding sequences forthe Fab molecules can also be produced synthetically using knownmethods. Nucleotide sequences can be designed with the appropriatecodons for the particular amino acid sequence desired. In general, onewill select preferred codons for the intended host in which thesequences will be expressed. The complete sequences are generallyassembled from overlapping oligonucleotides prepared by standard methodsand assembled into complete coding sequences. See, e.g., Edge (1981)Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al.(1984) J. Biol. Chem. 259:6311.

Expression Systems

Once the coding sequences for the heavy and light chain portions of theFab molecules are isolated or synthesized, they can be cloned into anysuitable vector or replicon for expression, for example, bacterial,mammalian, yeast and viral expression systems can be used. Numerouscloning vectors are known to those of skill in the art and are describedbelow. The selection of an appropriate cloning vector is a matter ofchoice.

i. Expression in Bacterial Cells

Bacterial expression systems can be used to produce the Fab molecules.Control elements for use in bacterial systems include promoters,optionally containing operator sequences, and ribosome binding sites.Useful promoters include sequences derived from sugar metabolizingenzymes, such as galactose, lactose (lac) and maltose. Additionalexamples include promoter sequences derived from biosynthetic enzymessuch as tryptophan (trp), the β-lactamase (bla) promoter system,bacteriophage λPL, and T7. In addition, synthetic promoters can be used,such as the tac promoter. The β-lactamase and lactose promoter systemsare described in Chang et al., Nature (1978) 275:615, and Goeddel etal., Nature (1979) 281: 544; the alkaline phosphatase, tryptophan (trp)promoter system are described in Goeddel et al., Nucleic Acids Res.(1980) 8:4057 and EP 36,776 and hybrid promoters such as the tacpromoter is described in U.S. Pat. No. 4,551,433 and deBoer et al.,Proc. Natl. Acad. Sci. USA (1983) 80:21-25. However, other knownbacterial promoters useful for expression of eukaryotic proteins arealso suitable. A person skilled in the art would be able to operablyligate such promoters to the Fab molecules for example, as described inSiebenlist et al., Cell (1980) 20:269, using linkers or adapters tosupply any required restriction sites. Promoters for use in bacterialsystems also generally contain a Shine-Dalgarno (SD) sequence operablylinked to the DNA encoding the Fab molecule. For prokaryotic host cellsthat do not recognize and process the native polypeptide signalsequence, the signal sequence can be substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria.

The foregoing systems are particularly compatible with Escherichia coli.However, numerous other systems for use in bacterial hosts includingGram-negative or Gram-positive organisms such as Bacillus spp.,Streptococcus spp., Streptomyces spp., Pseudomonas species such as P.aeruginosa, Salmonella typhimurium, or Serratia marcescans, amongothers. Methods for introducing exogenous DNA into these hosts typicallyinclude the use of CaCl₂ or other agents, such as divalent cations andDMSO. DNA can also be introduced into bacterial cells byelectroporation, nuclear injection, or protoplast fusion as describedgenerally in Sambrook et al. (1989), cited above. These examples areillustrative rather than limiting. Preferably, the host cell shouldsecrete minimal amounts of proteolytic enzymes. Alternatively, in vitromethods of cloning, e.g., PCR or other nucleic acid polymerasereactions, are suitable.

Prokaryotic cells used to produce the Fab molecules of this inventionare cultured in suitable media, as described generally in Sambrook etal., cited above.

ii. Expression in Yeast Cells

Yeast expression systems can also be used to produce the subject Fabmolecules. Expression and transformation vectors, eitherextrachromosomal replicons or integrating vectors, have been developedfor transformation into many yeasts. For example, expression vectorshave been developed for, among others, the following yeasts:Saccharomyces cerevisiae, as described in Hinnen et al., Proc. Natl.Acad. Sci. USA (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163;Candida albicans as described in Kurtz et al., Mol. Cell. Biol. (1986)6:142; Candida maltosa, as described in Kunze et al., J. BasicMicrobiol. (1985) 25:141; Hansenula polymorpha, as described in Gleesonet al., J. Gen. Microbiol. (1986) 132:3459 and Roggenkamp et al., Mol.Gen. Genet. (1986) 202:302; Kluyveromyces fragilis, as described in Daset al., J. Bacteriol. (1984) 158:1165; Kluyveromyces lactis, asdescribed in De Louvencourt et al., J. Bacteriol. (1983) 154:737 and Vanden Berg et al., Bio/Technology (1990) 8:135; Pichia guillerimondii, asdescribed in Kunze et al., J. Basic Microbiol. (1985) 25:141; Pichiapastoris, as described in Cregg et al., Mol. Cell. Biol. (1985) 5:3376and U.S. Pat. Nos. 4,837,148 and 4,929,555; Schizosaccharomyces pombe,as described in Beach and Nurse, Nature (1981) 300:706; and Yarrowialipolytica, as described in Davidow et al., Curr. Genet. (1985) 10:380and Gaillardin et al., Curr. Genet. (1985) 10:49, Aspergillus hosts suchas A. nidulans, as described in Ballance et al., Biochem. Biophys. Res.Commun. (1983) 112:284-289; Tilburn et al., Gene (1983) 26:205-221 andYelton et al., Proc. Natl. Acad. Sci. USA (1984) 81:1470-1474, and A.niger, as described in Kelly and Hynes, EMBO J. (1985) 4:475479;Trichoderma reesia, as described in EP 244,234, and filamentous fungisuch as, e.g, Neurospora, Penicillium, Tolypocladium, as described in WO91/00357.

Control sequences for yeast vectors are known and include promoterregions from genes such as alcohol dehydrogenase (ADH), as described inEP 284,044, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase(PyK), as described in EP 329,203. The yeast PHO5 gene, encoding acidphosphatase, also provides useful promoter sequences, as described inMyanohara et al., Proc. Natl. Acad. Sci. USA (1983) 80:1. Other suitablepromoter sequences for use with yeast hosts include the promoters for3-phosphoglycerate kinase, as described in Hitzeman et al., J. Biol.Chem. (1980) 255:2073, or other glycolytic enzymes, such as pyruvatedecarboxylase, triosephosphate isomerase, and phosphoglucose isomerase,as described in Hess et al., J. Adv. Enzyme Reg. (1968) 7:149 andHolland et al., Biochemistry (1978) 17:4900. Inducible yeast promotershaving the additional advantage of transcription controlled by growthconditions, include from the list above and others the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism,metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EP 073,657. Yeast enhancers also are advantageously used with yeastpromoters. In addition, synthetic promoters which do not occur in naturealso function as yeast promoters. For example, upstream activatingsequences (UAS) of one yeast promoter may be joined with thetranscription activation region of another yeast promoter, creating asynthetic hybrid promoter. Examples of such hybrid promoters include theADH regulatory sequence linked to the GAP transcription activationregion, as described in U.S. Pat. Nos. 4,876,197 and 4,880,734. Otherexamples of hybrid promoters include promoters which consist of theregulatory sequences of either the ADH2, GAL4, GAL10, or PHO5 genes,combined with the transcriptional activation region of a glycolyticenzyme gene such as GAP or PyK, as described in EP 164,556. Furthermore,a yeast promoter can include naturally occurring promoters of non-yeastorigin that have the ability to bind yeast RNA polymerase and initiatetranscription.

Other control elements which may be included in the yeast expressionvectors are terminators, for example, from GAPDH and from the enolasegene, as described in Holland et al., J. Biol. Chem. (1981) 256:1385,and leader sequences which encode signal sequences for secretion. DNAencoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene as describedin EP 012,873 and JP 62,096,086 and the α-factor gene, as described inU.S. Pat. Nos. 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO89/02463. Alternatively, leaders of non-yeast origin, such as aninterferon leader, also provide for secretion in yeast, as described inEP 060,057.

Methods of introducing exogenous DNA into yeast hosts are well known inthe art, and typically include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationsinto yeast can be carried out according to the method described in VanSolingen et al., J. Bact. (1977) 130:946 and Hsiao et al., Proc. Natl.Acad. Sci. USA (1979) 76:3829. However, other methods for introducingDNA into cells such as by nuclear injection, electroporation, orprotoplast fusion may also be used as described generally in Sambrook etal., cited above.

For yeast secretion the native polypeptide signal sequence may besubstituted by the yeast invertase, α-factor, or acid phosphataseleaders. The origin of replication from the 2μ plasmid origin issuitable for yeast. A suitable selection gene for use in yeast is thetrp1 gene present in the yeast plasmid described in Kingsman et al.,Gene (1979) 7:141 or Tschemper et al., Gene (1980) 10:157. The trp1 geneprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan. Similarly, Leu2-deficient yeast strains(ATCC 20,622 or 38,626) are complemented by known plasmids bearing theLeu2 Gene.

For intracellular production of the present polypeptides in yeast, asequence encoding a yeast protein can be linked to a coding sequence forthe Fab molecule to produce a fusion protein that can be cleavedintracellularly by the yeast cells upon expression. An example, of sucha yeast leader sequence is the yeast ubiquitin gene.

iii. Expression in Insect Cells

The Fab molecules can also be produced in insect expression systems. Forexample, baculovirus expression vectors (BEVs) are recombinant insectviruses in which the coding sequence for a foreign gene to be expressedis inserted behind a baculovirus promoter in place of a viral gene,e.g., polyhedrin, as described in Smith and Summers, U.S. Pat. No.4,745,051.

An expression construct herein includes a DNA vector useful as anintermediate for the infection or transformation of an insect cellsystem, the vector generally containing DNA coding for a baculovirustranscriptional promoter, optionally but preferably, followed downstreamby an insect signal DNA sequence capable of directing secretion of adesired protein, and a site for insertion of the foreign gene encodingthe foreign protein, the signal DNA sequence and the foreign gene beingplaced under the transcriptional control of a baculovirus promoter, theforeign gene herein being the coding sequence of the Fab molecule.

The promoter for use herein can be a baculovirus transcriptionalpromoter region derived from any of the over 500 baculoviruses generallyinfecting insects, such as, for example, the Orders Lepidoptera,Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example,but not limited to the viral DNAs of Autographo californica MNPV, Bombyxmori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleriamellonella MNPV, Aedes aegypti, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni. Thus, the baculovirus transcriptionalpromoter can be, for example, a baculovirus immediate-early gene IEI orIEN promoter; an immediate-early gene in combination with a baculovirusdelayed-early gene promoter region selected from the group consisting ofa 39K and a HindIII fragment containing a delayed-early gene; or abaculovirus late gene promoter. The immediate-early or delayed-earlypromoters can be enhanced with transcriptional enhancer elements.

Particularly suitable for use herein is the strong polyhedrin promoterof the baculovirus, which directs a high level of expression of a DNAinsert, as described in Friesen et al. (1986) “The Regulation ofBaculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES(W. Doerfler, ed.); EP 127,839 and EP 155,476; and the promoter from thegene encoding the p10 protein, as described in Vlak et al., J. Gen.Virol. (1988) 69:765-776.

The plasmid for use herein usually also contains the polyhedrinpolyadenylation signal, as described in Miller et al., Ann. Rev.Microbiol. (1988) 42:177 and a procaryotic ampicillin-resistance (amp)gene and an origin of replication for selection and propagation in E.coli. DNA encoding suitable signal sequences can also be included and isgenerally derived from genes for secreted insect or baculovirusproteins, such as the baculovirus polyhedrin gene, as described inCarbonell et al., Gene (1988) 73:409, as well as mammalian signalsequences such as those derived from genes encoding human α-interferonas described in Maeda et al., Nature (1985) 315:592-594; humangastrin-releasing peptide, as described in Lebacq-Verheyden et al., Mol.Cell. Biol. (1988) 8:3129; human IL-2, as described in Smith et al.,Proc. Natl. Acad. Sci. USA (1985) 82:8404; mouse IL-3, as described inMiyajima et al., Gene (1987) 58:273; and human glucocerebrosidase, asdescribed in Martin et al., DNA (1988) 7:99.

Numerous baculoviral strains and variants and corresponding permissiveinsect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori host cells have beenidentified and can be used herein. See, for example, the description inLuckow et al., Bio/Technology (1988) 6:47-55, Miller et al., in GENETICENGINEERING (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing,1986), pp. 277-279, and Maeda et al., Nature (1985) 315:592-594. Avariety of such viral strains are publicly available, e.g., the L-1variant of Autographa californica NPV and the Bm-5 strain of Bombyx moriNPV. Such viruses may be used as the virus for transfection of hostcells such as Spodoptera frugiperda cells.

Other baculovirus genes in addition to the polyhedrin promoter may beemployed in a baculovirus expression system. These includeimmediate-early (alpha), delayed-early (beta), late (gamma), or verylate (delta), according to the phase of the viral infection during whichthey are expressed. The expression of these genes occurs sequentially,probably as the result of a “cascade” mechanism of transcriptionalregulation. Thus, the immediate-early genes are expressed immediatelyafter infection, in the absence of other viral functions, and one ormore of the resulting gene products induces transcription of thedelayed-early genes. Some delayed-early gene products, in turn, inducetranscription of late genes, and finally, the very late genes areexpressed under the control of previously expressed gene products fromone or more of the earlier classes. One relatively well definedcomponent of this regulatory cascade is IEI, a preferred immediate-earlygene of Autographo californica nuclear polyhedrosis virus (AcMNPV). IEIis pressed in the absence of other viral functions and encodes a productthat stimulates the transcription of several genes of the delayed-earlyclass, including the preferred 39K gene, as described in Guarino andSummers, J. Virol. (1986) 57:563-571 and J. Virol. (1987) 61:2091-2099as well as late genes, as described in Guanno and Summers, Virol. (1988)162:444-451.

Immediate-early genes as described above can be used in combination witha baculovirus gene promoter region of the delayed-early category. Unlikethe immediate-early genes, such delayed-early genes require the presenceof other viral genes or gene products such as those of theimmediate-early genes. The combination of immediate-early genes can bemade with any of several delayed-early gene promoter regions such as 39Kor one of the delayed-early gene promoters found on the HindIII fragmentof the baculovirus genome. In the present instance, the 39 K promoterregion can be linked to the foreign gene to be expressed such thatexpression can be further controlled by the presence of IEI, asdescribed in L. A. Guarino and Summers (1986a), cited above; Guarino &Summers (1986b) J. Virol. (1986) 60:215-223, and Guarino et al. (1986c)J. Virol. (1986) 60:224-229.

Additionally, when a combination of immediate-early genes with adelayed-early gene promoter region is used, enhancement of theexpression of heterologous genes can be realized by the presence of anenhancer sequence in direct cis linkage with the delayed-early genepromoter region. Such enhancer sequences are characterized by theirenhancement of delayed-early gene expression in situations where theimmediate-early gene or its product is limited. For example, the hr5enhancer sequence can be linked directly, in cis, to the delayed-earlygene promoter region, 39K, thereby enhancing the expression of thecloned heterologous DNA as described in Guarino and Summers (1986a),(1986b), and Guarino et al. (1986).

The polyhedrin gene is classified as a very late gene. Therefore,transcription from the polyhedrin promoter requires the previousexpression of an unknown, but probably large number of other viral andcellular gene products. Because of this delayed expression of thepolyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEVsystem described by Smith and Summers in, for example, U.S. Pat. No.4,745,051 will express foreign genes only as a result of gene expressionfrom the rest of the viral genome, and only after the viral infection iswell underway. This represents a limitation to the use of existing BEVs.The ability of the host cell to process newly synthesized proteinsdecreases as the baculovirus infection progresses. Thus, gene expressionfrom the polyhedrin promoter occurs at a time when the host cell'sability to process newly synthesized proteins is potentially diminishedfor certain proteins such as human tissue plasminogen activator. As aconsequence, the expression of secretory glycoproteins in BEV systems iscomplicated due to incomplete secretion of the cloned gene product,thereby trapping the cloned gene product within the cell in anincompletely processed form.

While it has been recognized that an insect signal sequence can be usedto express a foreign protein that can be cleaved to produce a matureprotein, the present invention can also be practiced with a mammaliansignal sequence.

An exemplary insect signal sequence suitable herein is the sequenceencoding for a Lepidopteran adipokinetic hormone (AKH) peptide. The AKHfamily consists of short blocked neuropeptides that regulate energysubstrate mobilization and metabolism in insects. In a preferredembodiment, a DNA sequence coding for a Lepidopteran Manduca sexta AKHsignal peptide can be used. Other insect AKH signal peptides, such asthose from the Orthoptera Schistocerca gregaria locus can also beemployed to advantage. Another exemplary insect signal sequence is thesequence coding for Drosophila cuticle proteins such as CP1, CP2, CP3 orCP4.

Currently, the most commonly used transfer vector that can be usedherein for introducing foreign genes into AcNPV is pAc373. Many othervectors, known to those of skill in the art, can also be used herein.Materials and methods for baculovirus/insect cell expression systems arecommercially available in a kit form from companies such as Invitrogen(San Diego Calif.) (“MaxBac” kit). The techniques utilized herein aregenerally known to those skilled in the art and are fully described inSummers and Smith, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS ANDINSECT CELL CULTURE PROCEDURES, Texas Agricultural Experiment StationBulletin No. 1555, Texas A&M University (1987); Smith et al., Mol. Cell.Biol. (1983), and Luckow and Summers (1989). These include, for example,the use of pVL985 which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT, as described in Luckow and Summers, Virology (1989) 17:31.

Thus, for example, for insect cell expression of the presentpolypeptides, the desired DNA sequence can be inserted into the transfervector, using known techniques. An insect cell host can be cotransformedwith the transfer vector containing the inserted desired DNA togetherwith the genomic DNA of wild type baculovirus, usually bycotransfection. The vector and viral genome are allowed to recombineresulting in a recombinant virus that can be easily identified andpurified. The packaged recombinant virus can be used to infect insecthost cells to express a Fab molecule.

Other methods that are applicable herein are the standard methods ofinsect cell culture, cotransfection and preparation of plasmids are setforth in Summers and Smith (1987), cited above. This reference alsopertains to the standard methods of cloning genes into AcMNPV transfervectors, plasmid DNA isolation, transferring genes into the AcmMNPVgenome, viral DNA purification, radiolabeling recombinant proteins andpreparation of insect cell culture media. The procedure for thecultivation of viruses and cells are described in Volkman and Summers,J. Virol. (1975) 19:820-832 and Volkman et al., J. Virol. (1976)19:820-832.

iv. Expression in Mammalian Cells

Mammalian expression systems can also be used to produce the Fabmolecules. Typical promoters for mammalian cell expression include theSV40 early promoter, the CMV promoter, the mouse mammary tumor virus LTRpromoter, the adenovirus major late promoter (Ad MLP), and the herpessimplex virus promoter, among others. Other non-viral promoters, such asa promoter derived from the murine metallothionein gene, will also finduse in mammalian constructs. Mammalian expression may be eitherconstitutive or regulated (inducible), depending on the promoter.Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe Fab coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORYMANUAL, 2d edition, (Cold Spring Harbor Press, Cold Spring Harbor,N.Y.). Introns, containing splice donor and acceptor sites, may also bedesigned into the constructs of the present invention.

Enhancer elements can also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4: 761 and theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and human cytomegalovirus, as described inBoshart et al., Cell (1985) 41:521. A leader sequence can also bepresent which includes a sequence encoding a signal peptide, to providefor the secretion of the foreign protein in mammalian cells. Preferably,there are processing sites encoded between the leader fragment and thegene of interest such that the leader sequence can be cleaved either invivo or in vitro. The adenovirus tripartite leader is an example of aleader sequence that provides for secretion of a foreign protein inmammalian cells.

There exist expression vectors that provide for the transient expressionin mammalian cells of DNA encoding the Fab molecules. In general,transient expression involves the use of an expression vector that isable to replicate efficiently in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties. Once complete, the mammalian expressionvectors can be used to transform any of several mammalian cells. Methodsfor introduction of heterologous polynucleotides into mammalian cellsare known in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei. Generalaspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216. A synthetic lipidparticularly useful for polynucleotide transfection isN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, which iscommercially available under the name Lipofectin® (available from BRL,Gaithersburg, Md.), and is described by Felgner et al., Proc. Natl.Acad. Sci. USA (1987) 84:7413.

Mammalian cell lines available as hosts for expression are also knownand include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to, Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,Hep G2), human embryonic kidney cells, baby hamster kidney cells, mousesertoli cells, canine kidney cells, buffalo rat liver cells, human lungcells, human liver cells, mouse mammary tumor cells, as well as others.The mammalian host cells used to produce the Fab molecules of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM],Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium([DMEMl, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, or 4,560,655, WO 90/103430, WO87/00195, and U.S. Pat. No. RE 30,985, may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors such as insulin, transferrin, orepidermal growth factor, salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin(tm) M drug),trace elements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, Ph, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Preparing Specific Binding Molecules

Using the above techniques, a number of specific binding molecules thatexhibit immunological binding affinity for HCV E2 antigen can beprovided. In particular, depending on the expression system and hostselected, soluble Fab specific binding molecules can be readily producedby growing host cells transformed by an expression vector describedabove under conditions whereby the heavy and light chain portions areexpressed. Heterodimers comprising noncovalently associated heavy andlight chains can be isolated from the host cells and purified. Since thepresent invention also provides for the optional secretion of the heavyand light chain polypeptides, the Fab heterodimers can be purifieddirectly from the media. The selection of the appropriate growthconditions and recovery methods are within the skill of the art.

In addition, the Fab molecules of the present invention can be producedusing conventional methods of protein synthesis, based on theascertained amino acid sequences. In general, these methods employ thesequential addition of one or more amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected, under conditions that allow for the formation of an amidelinkage. The protecting group is then removed from the newly added aminoacid residue and the next amino acid (suitably protected) is then added,and so forth. After the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support,if solid phase synthesis techniques are used) are removed sequentiallyor concurrently, to render the final polypeptide. By simple modificationof this general procedure, it is possible to add more than one aminoacid at a time to a growing chain, for example, by coupling (underconditions which do not racemize chiral centers) a protected tripeptidewith a properly protected dipeptide to form, after deprotection, apentapeptide. See, e.g., J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984)and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis,Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, NewYork, (1980), pp. 3-254, for solid phase peptide synthesis techniques;and M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag,Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides:Analysis, Synthesis, Biology, supra, Vol. 1, for classical solutionsynthesis.

Recombinant human monoclonal antibody specific binding molecules can beprepared from the Fab molecules using known techniques. Bender et al.(1992) Hum Antibod Hybridomas 4:74. In particular, the coding sequencefor the heavy chain portion of a selected Fab clone can be inserted intoan expression vector along with the coding sequence for the constantdomains of a human Ig heavy chain, using the various recombinanttechniques described above. For example, the mammalian expression vectorpSG5 (Green et al. (1988) Nucleic Acids Res 16:369) can be used for thispurpose.

Cloning involves overlap PCR to remove the bacterial leader sequence(from the phagemid vector) and to modify the N-terminus of the heavychain coding sequence to a human consensus sequence. The coding sequencefor the light chain portion of the selected Fab clone can likewise beN-terminal modified to include a human consensus sequence, and clonedinto an expression vector such as PSG5. The PSG5 vectors contain an SV40origin of replication such that, on cotransfection of the heavy andlight chain vectors into mammalian cells, such as COS-7 cells,functional antibody molecule production can be confirmed. Burton et al.(1994) Science 266:1024-1027.

The heavy and light chains can subsequently be cloned into separatecloning vectors, and either the heavy or the light chain coding sequencesubcloned into the other vector to provide a combinatorial plasmid. Forexample, the heavy and light chain coding sequences can be respectivelyinserted into pEE6 and pEE12 vectors (Bebbington et al. (1992)Bio/Technology 10:169) which include a human cytomegalovirus promoterand the glutamine synthetase selectable marker. The heavy chain, alongwith control elements from the PEE6 vector can then be subcloned intothe PEE12 vector to provide a combinatorial plasmid. The combinatorialplasmid can be expressed in a CHO cell expression system. Those clonesfrom the CHO expression system which provide sufficient levels ofrecombinant antibody production can be selected for scale-up. Therecombinant antibodies expressed in the CHO-system can be purified usingknown techniques (e.g., affinity chromatography using protein A), andthe binding affinity of the recombinant specific binding moleculesassessed using an ELISA inhibition assay as described above.

Alternatively, the coding sequences for the Fab clones can betransferred into the vectors pcLCHC and pcIgG1, respectively, and thenexpressed as whole IgG in CHO cells as previously described. Samuelssonet al. (1996) Eur. J. Immunol. 26:3029.

Recombinant F(ab′)₂ and recombinant Fv specific binding molecules canalso be prepared from the phage-derived Fab clones using knowntechniques. Fv molecules generally comprise a non-covalently bound heavychain:light chain heterodimer which includes the antigen-binding portionof the Fab molecule and retains much of the antigen recognition andbinding capabilities of native antibody molecules. Inbar et al. (1972)Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976) Biochem15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096. Typically,the above-noted recombinant techniques used to construct the recombinantmonoclonal antibodies can be modified to provide the truncated specificbinding molecules. These molecules can also be cloned into CHOexpression systems, purified and characterized as above.

The phage-derived Fab clones can further be used to provide single chainFv (Sfv) molecules using known techniques. These Sfv molecules comprisea covalently linked heavy chain:light chain heterodimer which isexpressed from a gene fusion including the heavy and light chain codingsequences obtained from the phage-derived Fab molecule, wherein thechains are linked by a peptide-encoding linker. Huston et al. (1988)Proc. Nat. Acad. Sci. USA 85(16):5879-5883. A number of methods havebeen described to discern chemical structures for converting thenaturally aggregated—but chemically separated—heavy and light chainsinto an Sfv molecule which will fold into a three dimensional structuresubstantially similar to the structure of an antigen-binding site. See,e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S.Pat. No. 4,946,778, to Ladner et al.

In the practice of the invention, recombinant DNA design methods areused to develop appropriate chemical structures for linking the heavyand light chains into the Sfv binding molecule. Design criteria includedetermination of the appropriate length to span the distance between theC-terminus of one chain and the N-terminus of the other, wherein thelinker is generally formed from small hydrophilic amino acid residuesthat do not tend to coil or form secondary structures. Such methods havebeen described in the art. See, e.g., U.S. Pat. Nos. 5,091,513 and5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.

The first general step of linker design involves identification ofplausible sites to be linked. Appropriate linkage sites on each of theimmunoglobulin chains include those which will result in the minimumloss of residues from the heavy and light chains, and which willnecessitate a linker having a minimum number of residues consistent withthe need for molecule stability. A pair of sites defines a “gap” to belinked. Linkers connecting the C-terminus of one chain to the N-terminusof the next generally include hydrophilic amino acids which assume anunstructured configuration in physiological solutions and preferably arefree of residues having large side groups which might interfere withproper folding of the heavy and light chains. Thus, suitable linkerswould include polypeptide chains of alternating sets of glycine andserine residues, and may include glutamic acid and lysine residuesinserted to enhance solubility. One particular linker used in thepractice of the invention has the amino acid sequence [(Gly)₄Ser]₃.Another particularly preferred linker has the amino acid sequencecomprising 2 or 3 repeats of [(Ser)₄Gly], such as [(Ser)₄Gly]₃.Nucleotide sequences encoding such linker moieties can be readilyprovided using various oligonucleotide synthesis techniques known in theart. See, e.g., Sambrook, and Maniatis, supra.

Once the appropriate linker sequence has been ascertained, nucleotidesequences encoding the Sfv molecules can be joined using an overlap PCRapproach. See, e.g., Horton et al. (1990) BioTechniques 8:528-535. Theends of the light and heavy chains that are to be joined through theselected linker sequence are first extended by PCR amplification of eachchain, using primers that contain the terminal sequence of the chainregion followed by all or most of the desired linker sequence. Afterthis extension step, the light and heavy chains contain overlappingextensions which jointly contain the entire linker sequence, and whichcan be annealed at the overlap and extended by PCR to obtain thecomplete Sfv sequence using methods known in the art. Genes present inexpression cassettes comprising the sFv sequence can then be expressedin a suitable expression system, and the sFv molecules producedtherefrom can be purified and characterized as described above.

Vaccine Compositions

Therapeutic and prophylactic vaccine compositions are provided herein,which generally comprise mixtures of one or more of the above-describedanti-HCV monoclonal antibodies, including Fab molecules, Fv fragments,sFv molecules and combinations thereof. The prophylactic vaccines can beused to prevent HCV infection, and the therapeutic vaccines used totreat individuals following HCV infection. Prophylactic uses include theprovision of increased antibody titer to HCV in a vaccinated subject. Inthis manner, subjects at high risk of contracting HCV infection (e.g.,immunocompromised individuals, organ transplant patients, individualsobtaining blood or blood product transfusions, and individuals in closepersonal contact with HCV-infected individuals) can be provided withpassive immunity to the HCV agent. Furthermore, due to thecross-reactivity of the monoclonal antibodies, Fab molecules and sFvmolecules produced herein, a level of protection is afforded against anumber of heterologous HCV isolates. Other prophylactic uses for thepresent anti-HCV vaccines includes prevention of HCV disease in anindividual after exposure to the infectious agent. Therapeutic uses ofthe present vaccines involve both reduction and/or elimination of theinfectious agent from infected individuals, as well as the reductionand/or elimination of circulating HCV and the possible spread of thedisease.

The compositions can be administered in conjunction with ancillaryimmunoregulatory agents, for example, cytokines, lymphokines, andchemokines, including but not limited to IL-2, modified IL-2(cys125→ser125), GM-CSF, IL-12, γ-interferon, IP-10, MIP1β and RANTES.When the vaccine compositions are used as therapeutic vaccines, thecompositions can be administered in conjunction with known anti-HCVtherapeutics, such as α-interferon (α-IFN) therapy which generallyentails administration of 3 million units of α-IFN three times a weeksubcutaneously (Causse et al. (1991) Gastroenterology 101:497-502, Daviset al. (1989) N Engl J Med 321:1501-1506, Marcellin et al. (1991)Hepatology 13:393-397), interferon β (β-IFN) therapy (Omata et al.(1991) Lancet 338:914-915), ribivirin therapy (Di Bisceglie et al.(1992) Hepatology 16:649-654, Reichard et al. (1991) Lancet337:1058-1061) and antisense therapy (Wakita et al. (1994) J Biol Chem269:14205-14210). Therapeutic vaccine compositions comprising thepresent monoclonal antibodies can also be used in conjunction with knownanti-HCV combination therapies, for example, the combination of α-IFNand ursodiol (Bottelli et al. (1993) (Abstr.) Gastroenterology 104:879,O'Brien et al. (1993) (Abstr.) Gastroenterology 104:966) and thecombination of β-IFN and ribivirin (Kakumu et al. (1993)Gastroenterology 105:507-512).

The preparation of vaccine compositions containing one or moreantibodies, antibody fragments, sFv molecules or combinations thereof,as the active ingredient is generally known to those of skill in theart. Typically, such vaccines are prepared as injectables (e.g., eitheras liquid solutions or suspensions or as solid forms suitable forsolution or suspension in liquids prior to injection). The compositionswill generally also include one or more “pharmaceutically acceptableexcipients or vehicles” such as water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. Additionally, minoramounts of auxiliary substances, such as wetting or emulsifying agents,pH buffering substances, and the like, may be present in such vehicles.The vaccine compositions may be emulsified or the active ingredient(monoclonal antibodies) may be encapsulated in liposomes.

Once formulated, the vaccine compositions are conventionallyadministered parenterally, e.g., by injection (either subcutaneously orintramuscularly). Additional formulations suitable for other modes ofadministration include oral and pulmonary formulations, suppositories,and transdermal applications. For suppositories, traditional binders andcarriers may include, for example, polyalkylene glycols ortriglycerides. Such suppository formulations may be provided frommixtures containing the active ingredient(s) in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, and the like.

The vaccine compositions are administered to the subject to be treatedin a manner compatible with the dosage formulation, and in an amountthat will be prophylactically and/or therapeutically effective. Theamount of the composition to be delivered, generally in the range offrom 1 to 500 micrograms of active agent per dose, depends on thesubject to be treated, the capacity of the subject's immune system tomount its own immune-responses, and the degree of protection desired.The exact amount necessary will vary depending on the age and generalcondition of the individual to be treated, the severity of the conditionbeing treated and the particular anti-HCV agent selected and its mode ofadministration, among other factors. An appropriate effective amount canbe readily determined by one of skill in the art. Thus, a“therapeutically effective amount” of the composition will be sufficientto bring about treatment or prevention of HCV disease symptoms, and willfall in a relatively broad range that can be determined through routinetrials.

In addition, the vaccine compositions can be given in a single doseschedule, or preferably in a multiple dose schedule. A multiple doseschedule is one in which a primary course of vaccination may be with1-10 separate doses, followed by other doses given at subsequent timeintervals needed to maintain or reinforce the action of thecompositions. Thus, the dosage regimen will also, at least in part, bedetermined based on the particular needs of the subject to be treatedand will be dependent upon the judgement of the reasonably skilledpractitioner.

Gene Therapy

The recombinant monoclonal antibodies can also be used for gene therapy.In this regard, genes encoding the recombinant antibodies can beintroduced into a suitable mammalian host cell for expression orcoexpression using a number of viral based systems which have beendeveloped for gene transfer into mammalian cells. For example,retroviruses provide a convenient platform for gene delivery systems. Aselected nucleotide sequence encoding a V_(H) and/or a V_(L) domainpolypeptide can be inserted into a vector and packaged in retroviralparticles using techniques known in the art. The recombinant virus canthen be isolated and delivered to a subject. A number of suitableretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) HumanGene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns etal. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie andTemin (1993) Cur. Opin. Genet. Develop. 3:102-109. Particularlypreferred methods for producing and using retroviral vectors for genetherapy herein are described, for example, in International PublicationNo. WO 91/02805, published Mar. 7, 1991, and in U.S. patent applicationSer. No. 08/404,796, filed Mar. 15, 1995 for “Eukarotic Layered VectorInitiation Systems;” Ser. No. 08/405,627, filed Mar. 15, 1995 for“Recombinant α-Viral Vectors;” and Ser. No. 08/156,789, filed Nov. 23,1993 for “Packaging Cells.”

A number of suitable adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett etal. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human GeneTherapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al.(1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have been developedrecently for gene delivery. Such systems can include control sequences,such as promoter and polyadenylation sites, as well as selectablemarkers or reporter genes, enhancer sequences, and other controlelements which allow for the induction of transcription. AAV vectors canbe readily constructed using techniques well known in the art. See,e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International PublicationNos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (publishedMar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors which will find use for delivering the presentnucleic acid molecules encoding the Fab molecules include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing thegenes can be constructed as follows. The DNA encoding the particular Fabmolecule is first inserted into an appropriate vector so that it isadjacent to a vaccinia promoter and flanking vaccinia DNA sequences,such as the sequence encoding thymidine kinase (TK). This vector is thenused to transfect cells which are simultaneously infected with vaccinia.Homologous recombination serves to insert the vaccinia promoter plus thegene encoding the Fab molecule into the viral genome. The resulting TK⁻recombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the Fab molecules in ahost cell. In this system, cells are first infected in vitro with avaccinia virus recombinant that encodes the bacteriophage T7 RNApolymerase. This polymerase displays exquisite specificity in that itonly transcribes templates bearing T7 promoters. Following infection,cells are transfected with the polynucleotide of interest, driven by aT7 promoter. The polymerase expressed in the cytoplasm from the vacciniavirus recombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation products. See, e.g., Elroy-Steinand Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al.,Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the Fab-encoding nucleotide sequences. Theuse of an avipox vector is particularly desirable in human and othermammalian species since members of the avipox genus can onlyproductively replicate in susceptible avian species and therefore arenot infective in mammalian cells. Methods for producing recombinantavipoxviruses are known in the art and employ genetic recombination, asdescribed above with respect to the production of vaccinia viruses. See,e.g., the International Publications WO 91/12882; WO 89/03429, publishedApr. 20, 1989; and WO 92/03545, published Mar. 5, 1992.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 andWagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery under the invention.

Assay Reagents and Diagnostic Kits

The above-described anti-HCV binding molecules (the recombinantmonoclonal antibodies, including Fab molecules, Fv fragments and sFvmolecules) which are capable of reacting immunologically with samplescontaining HCV particles are also used herein to detect the presence ofHCV viral particles and/or viral antigens in specific binding assays ofbiological samples. In particular, the novel specific binding moleculesof the present invention can be used in highly sensitive methods forscreening and identifying individuals carrying and/or infected with HCV,as well as for screening for HCV-contaminated blood or blood products.The present binding molecules can also be used in assays for monitoringthe progress of anti-HCV therapies in treated individuals, and formonitoring the growth rate of HCV cultures used in research andinvestigation of the HCV agent.

The format of specific binding assays will be subject to a great deal ofvariation in accordance with procedures that are well known in the art.For example, specific binding assays can be formatted to utilize one, ora mixture of several, of the recombinant human monoclonal antibodies,(including Fab molecules, Fv fragments as well as sFv molecules) thathave been prepared according to the present invention. The assay formatcan be generally based, for example, upon competition, direct bindingreaction or sandwich-type assay techniques. Furthermore, the presentassays can be conducted using immunoprecipitation or other techniques toseparate assay reagents during, or after commencement of, the assay.Other assays can be conducted using specific binding molecules that havebeen insolubilized prior to commencement of the assay. In this regard, anumber of insolubilization techniques are well known in the art,including, without limitation, insolubilization by adsorption to animmunoadsorbant or the like, absorption by contact with the wall of areaction vessel, covalent crosslinking to insoluble matrices or “solidphase” substrates, noncovalent attachment to solid phase substratesusing ionic or hydrophobic interactions, or by aggregation usingprecipitants such as polyethylene glycol or cross-linking agents such asglutaraldehyde.

There are a large number of solid phase substrates which can be selectedfor use in the present assays by those skilled in the art. For example,latex particles, microparticles, magnetic-, para-magnetic- ornonmagnetic-beads, membranes, plastic tubes, walls of microtitre wells,glass or silicon particles and sheep red blood cells all are suitablefor use herein.

In general, most of the present assays involve the use of a labeledbinding complex formed from the combination of a specific bindingmolecule (recombinant monoclonal antibodies, Fab fragments, Fv fragmentsand sFv molecules) with a detectable label moiety. A number of suchlabels are known in the art and can be readily attached (either usingcovalent or non-covalent association techniques) to the bindingmolecules of the present invention to provide a binding complex for usein the above-noted assay formats. Suitable detectable moieties include,but are not limited to, radioactive isotopes, fluorescers, luminescentcompounds (e.g., fluorescein and rhodamine), chemiluminescers (e.g.,acridinium, phenanthridinium and dioxetane compounds), enzymes (e.g.,alkaline phosphatase, horseradish peroxidase and beta-galactosidase),enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, and metalions. These labels can be associated with the binding molecules usingattachment techniques that are known in the art.

Exemplary assay methods generally involve the steps of: (1) preparingthe detectably labeled binding complexes as above; (2) obtaining asample suspected of containing HCV particles and/or HCV antigen; (3)incubating the sample with the labeled complexes under conditions whichallow for the formation of a specific binding molecule-antigen complex(e.g., an antibody-antigen complex); and (4) detecting the presence orabsence of labeled binding molecule-antigen complexes. As will beappreciated by those skilled in the art upon the reading of thisspecification, such assays can be used to screen for the presence of HCVinfection in human donor blood and serum products, for monitoring thegrowth rate of HCV cultures in diagnostic and/or research settings, fordetecting HCV infection in an individual, or for monitoring thetherapeutic effect of an anti-HCV treatment protocol in an infectedsubject. When the assays are used in the clinical setting, e.g., fordetecting HCV infection or monitoring anti-HCV therapies, samples can beobtained from human and animal body fluids, such as whole blood, serum,plasma, cerebrospinal fluid, urine and the like. Furthermore, the assayscan be readily used to provide quantitative information using referenceto standards or calibrants as known in the art.

In one particular assay method of the invention, an enzyme-linkedimmunosorbent assay (ELISA) can be used to quantify an HCV antigenconcentration in a sample. In the method, the specific binding moleculesof the present invention are conjugated to an enzyme to provide alabeled binding complex, wherein the assay uses the bound enzyme as aquantitative label. In order to measure antigen, a binding moleculecapable of specifically binding the selected HCV antigen (e.g., anantibody molecule) is immobilized to a solid phase substrate (e.g., amicrotitre plate or plastic cup), incubated with test sample dilutions,washed and incubated with the binding molecule-enzyme complexes of theinvention, and then washed again. In this regard, suitable enzyme labelsare generally known, including, for example, horseradish peroxidase.Enzyme activity bound to the solid phase is measured by adding thespecific enzyme substrate, and determining product formation orsubstrate utilization colorimetrically. The enzyme activity bound to thesolid phase substrate is a direct function of the amount of antigenpresent in the sample.

In another particular assay method of the invention, the presence of HCVin a biological sample (e.g., as an indicator of HCV infection) can bedetected using strip immunoblot assay (SIA) techniques, such as thoseknown in the art which combine traditional Western and dot blottingtechniques, e.g., the RIBA® (Chiron Corp., Emeryville, Calif.) test. Inthese assays, one or more of the specific binding molecules (therecombinant monoclonal antibodies, including Fab molecules) areimmobilized as individual, discrete bands on a membranous support teststrip. Visualization of reactivity with HCV particles present in thebiological sample is accomplished using sandwich binding techniques withlabeled antibody-conjugates in conjunction with a colorimetric enzymesubstrate. Internal controls can also be present on the strip. The assaycan be performed manually or used in an automated format.

Furthermore, the recombinant human monoclonal antibodies, (including Fabmolecules, Fv fragments as well as sFv molecules) that have beenprepared according to the present invention can be used in affinitychromatography techniques in order to detect the presence of HCV in abiological sample. Such methods are well known in the art.

Kits suitable for use in conducting any of the above-described assaysand affinity chromatography techniques, and containing appropriatelabeled binding molecule complex reagents can also be provided inaccordance with the practice of the invention. Assay kits are assembledby packaging the appropriate materials, including all reagents andmaterials necessary for conducting the assay in a suitable container,along with an appropriate set of assay instructions.

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Experimental EXAMPLE 1 Characterization of the Library Donor

Bone marrow was obtained from a 60 year old asymptomatic, male blooddonor, who was found to be HCV positive during regular screening inconjunction with a blood donation. The cause of infection was unknown.The donor was unimmunized, and had received no treatment for the HCVinfection prior to the bone marrow aspiration, which amounted toapproximately 3 ml. The genotype of HCV in the donor's serum at the timeof bone marrow donation was determined using a published method andfound to be HCV 2b. Widell et al. (1994) J Med Virol 44:272-279.

In particular, the nucleotide sequence of the hypervariable region 1(HVR1) of the E2 gene of the HCV isolate from the donor was obtained asfollows. Single stranded template DNA was obtained, and a sequencingreaction was performed using newly designed primers (A. Widell et al,manuscript in preparation), in a cycle sequencing reaction with labellednucleotides (PCR cycle sequencing kit, Perkin-Elmer) according to themanufacturers instructions. Allander et al. (1994) J Med Virol43:415-419. The reaction product was run on an automated sequencer(Applied Biosystems, Calif.), and the data edited and analyzed usingMacMolly software (available from SoftGene, Berlin, Germany). Thededuced amino acid sequence of the HVR-E2 region of the donor isolatewas determined to be as follows:VAGVDASTYTTGGQSGRTTYGIVGLFSLGPSQKLSLINTNGSWHINR (SEQ ID NO:28).

EXAMPLE 2 Construction of the Phage Display Library

Lymphocytes were isolated from the bone marrow sample obtained inExample 1 using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). TotalRNA was extracted by the acid phenol extraction method (Chomczynski etal. (1987) Anal Biochem 162:156-159), first strand cDNA synthesisutilizing oligo-dT priming of 10 μg of RNA was performed (cDNA synthesiskit, Pharmacia Biotech) and heavy (Fd) and light chain DNA was PCRamplified using 5′ biotinylated primers of previously publishedsequences for γ1 heavy and κ light chains (5′ primers: VH1a, VH1f, VH2f,VH3a, VH3f, VH4f, VH6a, VH6f, Vk1a, Vk2a, Vk3a; 3′-primers: CG1z andCK1a) (available from Scandinavian Gene Synthesis, Köping, Sweden). See,e.g., Persson et al. (1991) Proc Natl Acad Sci USA 88:2432-2436, Kang etal. (1991) Methods: Comp. Meth Enzymol 2:111-118.

PCR was performed using a Thermal Cycler 4800 (Perkin-Elmer) at 94° C.for 5 minutes, then 35 cycles of 940 for 60 seconds, 520 for 30 seconds,and 720 for 180 seconds. After the 35 cycles, an extension step at 720for 10 minutes completed the PCR procedure. PCR products were analyzedby running a fraction of each in a 1.5% agarose gel, the heavy and lightchain DNA were pooled separately, phenol-chloroform extracted and thenethanol precipitated.

12 μg each of heavy and light chain DNA was gel purified on a 2.5%agarose gel, electroeluted (Schleicher & Schuell, Germany), and digestedwith the restriction endonucleases XhoI/SpeI, and SacI/XbaI,respectively (available from Life Technologies, Gaithersburg, Md.). Thedigested PCR products were subsequently gel purified, recovered byelectroelution, and ligated into the vector pComb3H (Barbas, III et al.(1995) Methods: Comp. Meth Enzymol 8:94-103) after it had been digestedwith the corresponding restriction enzymes and gelpurified/electroeluted as previously described for similar vectorsystems (Yang et al. (1995) J Mol Biol 254:392-403, Barbas, III et al.(1991) Proc Natl Acad Sci USA 88:7978-7982). For ligations, T4 ligase(Life Technologies) was used at 0.5 units per 10 μl reaction volume atapproximately 16° C. over night. The combinatorial library was expressedon phage, including harvesting of phage, as reported. Samuelsson et al.(1995) Virology 207:495-502.

Ligation with light chain genes into pComb3H gave a library of 2×10⁷members. The subsequent ligation of Fd genes into this library resultedin a γ1/κ library with 2×10⁶ members.

EXAMPLE 3 Preparation of the HCV E2 Selecting Antigens

A truncated, secreted form of the HCV E2 molecule was constructed whichincludes amino acids 383_(ala) through 715_(lys) (using the nomenclatureof Choo et al. (1991) Proc Natl Acad Sci USA 88:2451-2455). The E2molecule was expressed using a Chinese hamster ovary cell/dihydrofolatereductase (CHO/DHFR) expression system to provide a “conformational HCVE2 antigen” as follows. A DNA fragment of HCV E2 from amino acid 383 toamino acid 715 of HCV1 was generated by PCR and then ligated into aplasmid vector having the murine cytomegalovirus (MCMV) immediate earlypromoter/enhancer (Dorsch-Hasler et al. (1985) Proc Natl Acad Sci USA82:8325-8329) and the selectable dhfr gene marker. The resultant plasmidwas then stably transfected into dhfr⁻ CHO cells to generate a stablerecombinant CHO cell line which secreted the conformational HCV E2antigen.

The conformational E2 antigen was purified using known methods (Rosa etal. (1996) Proc Natl Acad Sci USA 93:1759-1763) as follows. Conditionedmedia from the CHO cells was concentrated 15-fold by ultrafiltration,followed by a further 10-fold volume reduction by ammonium sulfateprecipitation at 75% saturation, and redissolution into 25 mM Trischloride/1 mM EDTA, pH 7.5. The monoclonal antibody 5E5/H7 (raisedagainst HeLa E1/E2) was used for purification. The antibody column wasequilibrated in 25 mM Tris chloride/0.15 M NaCl, pH 7.5. The ammoniumsulfate-precipitated E2 was dissolved in 25 mM Tris chloride/1 mM EDTA,pH 7.5, and loaded onto the column. The column was washed with phosphatebuffered saline (PBS)/1 M NaCl and then eluted with 3-4 column volumesof Actisep (Sterogene, Arcadia, Calif.). All of the yellow-coloredActisep-containing fractions were pooled, concentrated in a stirred cellultrafilter, and diafiltered into PBS buffer.

A recombinant HCV E1/E2 complex antigen was constructed and expressedusing a Chinese hamster ovary cell/dihydrofolate reductase (CHO/DHFR)expression system as follows. Plasmid pMCMV-HC5p (Spaete et al. (1992)Virology 188:819-830) which encodes the HCV structural region as a 917amino acid polypeptide spanning Met₁ to Gly₉₁₇ of the HCV1 genome wasgenerated by cloning a 2813 base pair (bp) HCV StuI fragment from pGEM-4blue-HC5p-1 8 into the unique SalI site of the mammalian cell expressionvector pMCVAdhfr (Spaete et al. (1990) J Virol 64:2922-2931). ThepMCVAdhfr vector encodes the selectable dhfr gene with transcription ofthe expressed gene driven by the MCMV immediate early promoter/enhancerand terminated by SV40 polyadenylation sequences. The Klenow fragment(Boehringer-Mannheim Biochemicals, Indianapolis, Ind.) was used to fillthe SalI site prior to ligation of the StuI fragment. The pMCMV-HC5pplasmid was stably transfected into dhfr⁻ CHO cells to generate a stablerecombinant CHO cell line (#62) which expresses the recombinant HCVE1/E2 complex antigen.

The recombinant E1/E2 complex antigen was purified using known methods(Choo et al. (1994) Proc Natl Acad Sci USA 91:1294-1298) as follows. CHOcell line #62 was harvested by pelleting and freezing. After lysis byDounce homogenization in hypotonic buffer, the pellet was extracted byhomogenizing in 2% Triton X-100®, and E1 (33 kD) and E2 (72 kD) wereselectively copurified by successive chromatography on agarose-boundGralanthus Nivalis-lectin (Vector Laboratories) and fast-flowS-Sepharose cation exchanger (Pharmacia).

EXAMPLE 4 Panning of the Combinatorial Library

Antigenic selection of specifically-binding Fab molecules was conductedusing a variation on known techniques. Burton et al. (1991) Proc NatlAcad Sci USA 88:10134-10137. In particular, four wells of a microtiterplate (Costar 3690, Cambridge, Mass.) were coated at 4° C. overnightwith 50 μl of either purified recombinant conformational HCV E2 antigenor purified recombinant E1/E2 complex antigen (prepared in Example 3),expressed in CHO cells, at 2.5 μg/ml. Spaete et al. (1992) Virology188:819-830. Blocking was effected by completely filling the wells with5% non-fat dry milk in PBS for 1 h at 22° C. 50 Al of the phage library(5×10¹⁰ cfu) was added to each well, and the plate was incubated for 2hours at 37° C. Phage were removed and each well was washed bycompletely filling with a solution of PBS and 0.5% Tween 20 for 5minutes, and then thoroughly removing the wash solution. Washing wasperformed 1-10 times as described below. Phage were eluted by adding 50μl per well of elution buffer (0.1M HCl adjusted to pH 2.2 with solidglycine) and incubating for 10 minutes at ambient temperature. Theelution buffer was removed and neutralized with 3 μl of 2M Tris base per50 μl of elution buffer. E. coli XL-1 blue cells (Barbas, III et al.(1991) Methods: Comp. Meth Enzymol 2:119-124) were infected by theeluted phage, aliquots plated, and propagation of phage after each roundof panning effected as has been described. Samuelsson et al. (1995)Virology 207:495-502.

In the first group of pannings (Panning Series I), the number ofwashings was increased in each subsequent panning round for three rounds(1, 3 and 10 washings, respectively), while in the second group ofpannings (Panning Series II), a single panning round with 10 washingswas performed. As can be seen by the results depicted in Table I, a 100fold increase in eluted phage was noted in Panning Series I.

TABLE I Washes Eluted Phage Enrichment Panning No. (No.) (cfu) FactorPanning Series I 1  1 2.6 × 10⁶ — 2  3 2.3 × 10⁷  9 3 10 2.6 × 10⁸ 100Panning Series II 1 10 2.4 × 10⁵

The antigen used in both the Series I and II pannings was therecombinant conformational E2 antigen. A third group of pannings(Panning Series III) was performed as in Series I; however, therecombinant E1/E2 complex antigen was used to select positive clones.

EXAMPLE 5 Expression of the Fab Molecules

Fab molecules were expressed by growing ampicillin resistant E. coliXL-1 blue cell colonies, containing Fab plasmids, with the gIII gene(encoding the cpIII anchor protein) (a) intact to provide insoluble Fabfragments, or (b) deleted by digestion with SpeI and NheI to providesoluble Fab fragments (digestion with these enzymes provides compatiblecohesive ends; thus, the resulting DNA fragment lacking the gIIIfragment can be gel-purified and self-ligated), in SB medium (superbroth; 30 g tryptone, 20 g of yeast extract, and 10 g of MOPS per liter,pH 7) (Burton et al. (1991) Proc Natl Acad Sci USA 88:10134-10137)containing 50 μg/ml ampicillin and 1% glucose, until an OD_(600 nm) ofabout 1.0 was reached. The bacterial host cells were pelleted bycentrifugation, and media exchanged to SB medium with 1 mM IPTG and 20mM MgCl₂, and the cells resuspended. The resulting culture was incubatedat room temperature on a shaker platform set at about 290 rpm and leftovernight. The following day, cells were spun down, the supernatantdiscarded, PBS added (to between 2 to 4% of original culture volume) andthe periplasmic contents of the bacterial cells released by three cyclesof freeze-thawing. Bacterial debris was pelleted by centrifugation, andthe Fab molecule-containing supernatant aliquoted to new vials. The Fabmolecules were maintained at −20° C. until used.

EXAMPLE 6 Expression Levels of the Fab Clones

The expression levels of Fab molecules (obtained in Example 5) wereascertained using known ELISA techniques. Samuelsson et al. (1995)Virology 207:495-502. In particular, goat anti-human F(ab′)₂ (Pierce,USA) or goat anti-human Fd (The Binding Site, UK) was diluted 1:1000 in0.1 M carbonate-bicarbonate buffer, pH 9.6 and coated on microtiterwells by incubation overnight at 4° C. Coating solution was discarded,and the wells were blocked with 5% dry milk in PBS for 1 hr at ambienttemperature, after which the blocking solution was removed, and Fabsamples (from Example 5) at appropriate dilutions in PBS-T were added.After incubation at ambient temperature for 1 hour, the plates werewashed, and ALP-goat anti human F(ab′)₂ at a 1:500 dilution was added.After 1 hour incubation, and five subsequent washes, the label substratesolution, p-nitrophenylphosphate (Sigma, USA) in 0.1 M diethanolamine,pH 9.8 was added. Absorbance was measured at 405 nm in a microplatereader (Dynastar, Mass.). Most Fab clones were found to produce between0.2 and 2.0 mg Fab/L culture, corresponding to 10-100 μg/ml in theperiplasmic preparations. The expressed Fab clones were screened for E2reactivity and promptly sequenced, in order to identify multiple copiesof the same original clone.

EXAMPLE 7 Western Blot for Heavy and Light Chain Expression

In order to test for correct expression of both chains, several Fabmolecules were analyzed in Western blots using antiserum for human Fd-and light chains. In particular, 10 μl of the periplasmic Fab moleculepreparations (prepared in Example 5) were separated on a precast 12%Tris-glycine polyacrylamide gel, and transferred to a nitrocellulosemembrane by electroblotting using an Xcell Mini-cell apparatus (NovexExperimental Technology, San Diego, Calif.). The membrane was blocked in5% dry milk over night, and incubated with either alkaline phosphatasecoupled anti-human Fab antiserum (available from Pierce) diluted 1:1000in 5% dry milk and 0.05% Tween 20 (PBS-MT) for 3 h at 22° C. duringconstant rocking. The subject anti-human Fab antiserum was chosen sinceit is known to be mainly reactive to light chains. To detect the heavy(Fd) chain expression products, strips were first incubated with asheep-anti-human Fd serum (Binding Site, U.K.) which was diluted to1:1000 and incubated (as above), washed and then incubated again with asecondary antibody, AP-anti-goat IgG (Sigma, St. Louis, Mo.) at adilution of 1:500.

Following the last incubation for 1 hour at 22° C., the membranes werewashed three times in PBS-T, and color development was performed with 2ml BCIP/NBT solution (Sigma, St Louis, Mo.) for 7 minutes. Membraneswere rinsed in water and dried. Prestained molecular weight markers(Amersham, U.K.) were used in each blot.

For all clones tested, expression of both chains was approximatelyequivalent. The heavy chain (expressed as a fusion polypeptide with thetruncated gIII protein) showed an approximate molecular weight of 70 kD.

EXAMPLE 8 Sequencing of the Fab Clones

Plasmid DNA from each Fab molecule clone grown in the E. coli XL-1 bluecell cultures (in Example 5), was isolated using a Wizard mini prep DNApurification reagent system (Promega). Single stranded DNA was obtainedby PCR, using primers that hybridized upstream and downstream of thecloning regions in the pComb3H vector (pC3H-2488S: 5′-CAA CGC AAT TAATGT GAG TTA G (SEQ ID NO:29); G-back: 5′-GCC CCC TTA TTA GCG TTT GCC ATC(SEQ ID NO:30). In each reaction, one of the two PCR primers used wasbiotinylated at the 5′ terminus. After 35 cycles of PCR amplification,single stranded DNA was obtained by denaturing the DNA under alkalineconditions and absorbing the biotinylated DNA strand to streptavidincoated beads (Dynal, Oslo, Norway) using known techniques. Hultman etal. (1989) Nucl Acid Res 17:4937-4945.

Dideoxy sequencing reactions according to the method of Sanger et al.(1977) Proc Natl Acad Sci USA 74:5463-5467 was performed utilizingFITC-labelled primers hybridizing 3′ of the junction between thevariable and constant Ig regions or 5′ to the start of the heavy andlight chain genes. Particularly, SEQKb: 5′-ATA GAA GTT GTT CAG CAG GCA(SEQ ID NO:31) and omp-seq: 5′-AAG ACA GCT ATC GCG ATT GCA G (SEQ IDNO:32) were used for the κ light chains. SEQGb: 5′-GTC GTT GAC CAG GCAGCC CAG (SEQ ID NO:33) and pel-seq: 5′-ACC TAT TGC CTA CGG CAG CCG (SEQID NO:34) were used for the γ heavy chains. The reaction products wererun on an automated sequencer (A.L.F., Pharmacia Biotech), and weretranslated and aligned using the MacMolly software (SoftGene, Berlin,Germany).

From the first series of pannings conducted in Example 4 (Panning SeriesI), 10 Fab molecule clones (identified as Fab molecule clones L1-L10)that were assayed for expression (as described in Examples 6 and 7) weresequenced using the above-described sequencing method. These 10 cloneswere found to have very similar CDR3 sequences in their heavy chains(the H3 region), indicating that they all derived from the same B-cellclone. Litwin et al. (1990) J Exp Med 171:293-297. However, while theVDJ junctions and the length of the H3 regions were identical, a numberof different point mutations were identified in their heavy chains, andeach heavy chain was combined with a different light chain. Two clones,identified as Fab molecule clones L1 and L3, were selected for furthertesting.

From the second series of pannings conducted in Example 4 (PanningSeries II), 20 Fab molecule clones (identified as Fab molecule clones1:1-1:20) that were assayed for expression (as described in Examples 6and 7) were sequenced. From this round of sequencing, 6 Fab clones werefound to produce insufficient levels of Fab, and 4 Fab clones were foundto exhibit cross reactivity to control antigen in a specific bindingassay. From the sequencing information obtained from the remaining 10Fab clones, it was found that 7 Fab clones carried heavy chains relatedto the ones found in the Panning Series I (the L1-10 Fab moleculeclones). However, 3 of the 7 Fab clones had distinctly different H3regions from the Panning Series I clones, and were also unique relativeto each other. These clones were selected for further testing andidentified herein as Fab molecule clones 1:5, 1:7, 1:11.

From the third series of pannings conducted in Example 4 (Panning SeriesIII), 30 Fab molecule clones were assayed for expression (as describedin Examples 6 and 7), and 16 were found to be reactive to both the E1/E2complex antigen and to the E2 antigen alone. These 16 clones weresequenced as above. 12 of the 16 clones that were sequenced were foundto have a H3 sequence similar to clones L1-L10, while the remaining 4were found to have unique H3 sequences. Two of the 4 clones havingunique H3 sequences were selected for further testing and are identifiedherein as Fab molecule clones A8 and A12.

The κ light chain nucleic acid sequences of the following Fab moleculeclones: 1:5 (SEQ ID NO:15); 1:7 (SEQ ID NO:16); 1:11 (SEQ ID NO:17); L3(SEQ ID NO:18); L1 (SEQ ID NO:19); A8 (SEQ ID NO:20); and A12 (SEQ IDNO: 21) are depicted in FIGS. 3A-3G, respectively.

The γ1 heavy chain nucleic acid sequences of the following Fab moleculeclones: 1:5 (SEQ ID NO:22); 1:7 (SEQ ID NO:23); 1:11 (SEQ ID NO:24); L3(SEQ ID NO:25); L1 (SEQ ID NO:26); A8 (SEQ ID NO:27); and A12 (SEQ IDNO:28) are depicted in FIGS. 4A-4G, respectively.

The deduced γ1 heavy chain amino acid sequences of Fab molecule clones1:5 (SEQ ID NO:1); 1:7 (SEQ ID NO:2); 1:11 (SEQ ID NO:3); L3 (SEQ IDNO:4); L1 (SEQ ID NO:5); A8 (SEQ ID NO:6); and A12 (SEQ ID NO:7) aredepicted in FIGS. 1A-1G, respectively. The CDR regions (CDR1, CDR2 andCDR3) from each chain have been identified in the Figures.

The deduced κ light chain amino acid sequences of Fab molecule clones1:5 (SEQ ID NO:8); 1:7 (SEQ ID NO:9); 1:11 (SEQ ID NO:10); L3 (SEQ IDNO:11); L1 (SEQ ID NO:12); A8 (SEQ ID NO:13); and A12 (SEQ ID NO:14) aredepicted in FIGS. 2A-2G, respectively. The CDR regions (CDR1, CDR2 andCDR3) from each chain have also been identified as noted above.

In summary, out of 50 clones that were obtained from the three panningseries, 36 were found to be specific to E2, and 29 of those 36E2-specific clones share a related heavy chain.

EXAMPLE 9 ELISA Assay for HCV E2 Antigen Reactivity

The Fab molecule clones 1:5, 1:7, 1:11 and L3 were screened for HCV E2antigen reactivity as follows. Either recombinant conformational HCV E2antigen, or recombinant HCV E1/E2 complex antigen (prepared as describedin Example 3) was diluted to 0.25 μg/ml in 0.05 M carbonate-bicarbonatebuffer, pH 9.6, and coated to microtitre wells (Costar #3690; LifeTechnologies) overnight at 4° C. Unbound antigen was discarded, and thewells were blocked with 5% nonfat dry milk in PBS for 60 minutes atambient temperature. After the blocking solution was discarded,solutions containing the Fab molecules to be tested were added at 1:2,1:10 and 1:100 dilutions (diluent: PBS with 0.1% NP-40). The plates wereincubated at ambient temperature for 2 hours, washed five times with PBSwith 0.05% Tween 20 (PBS-T), and ALP-goat anti-human F(ab′)₂ (Pierce,Rocherford, Ill.) was added at a 1:1000 dilution. After 60 minutes andsubsequent washes, substrate solution (p-nitrophenylphosphate) (SIGMA,St. Louis, Mo.) was added and absorbance was measured at 405 nm in amicroplate reader (Dynastar, Mass.).

The cut-off value for positive readings was set at 4 times the OD valueobtained for a negative control sample which comprised an anti-HIV Fabof equal concentrations. Barbas III et al. (1991) Proc Natl Acad Sci USA88:7978-7982. For control purposes, bovine serum albumin (BSA) (SIGMA),HIV gp120_(LAI) (Intracell, Cambridge, Mass.) and tetanus toxoid (TT)(SBL Vaccin, Solna, Sweden) coated at 5, 1, and 1 μg/ml, respectively,were used in corresponding ELISAs as controls for unspecific reactivity.

The results from the ELISAs are depicted in Table II below. As can beseen, Fab molecules expressed from the 1:5, 1:7, 1:11 and L3 clones eachreacted strongly with both the conformational HCV E2 antigen and the HCVE1/E2 complex antigen, while showing no cross reaction with the controlantigens (BSA, HIVgp120 and TT).

TABLE II ELISA reactivity to: [Fab] HIV Clone (μg/ml)¹ E2² E1/E2² BSA²gp120² TT² L 3 100  1.695 2.460 0.089 ND 0.313 1:5  10  0.219 0.6140.031 ND   0.012³ 1:7 100 >3.000 >3.000  0.006 ND ND  1:11  10 >3.0001.831 0.506 ND ND ¹Fab concentration in the periplasmic preparation usedin the analyses. ²OD_(405 nm), sample diluted 1:10. ³sample diluted1:100.

EXAMPLE 10 Western Blot Assay for HCV E2 Antigen Reactivity

The Fab molecule clones 1:5, 1:7, 1:11 L1, L3, A8 and A12 were screenedfor HCV E2 antigen reactivity using the following techniques. Westernblots were conducted using 1 μg of the recombinant conformational HCV E2glycoprotein (obtained in Example 3) that was denatured by heating to98° C. for 5 min in a Laemmli buffer, separated on a 8-16%polyacrylamide gradient gel (Novex Experimental Technologies), andtransferred to a nitro-cellulose membrane that was blocked as describedabove, and cut into strips. Subsequently, each strip was incubated witha Fab preparation (expressed from the 1:5, 1:7, 1:11, L3, L1, A8 and A12clones) that was diluted 1:20 in PBS-MT for 2 hours at 22° C. withconstant rocking. The strips were washed three times in PBS-T, andalkaline phosphatase conjugated goat anti-human Fab serum (Pierce),diluted 1:1000 in PBS-MT, was added. Following incubation for 1 hour at22° C., the strips were again washed three times in PBS with 0.05% Tween20, and color development was performed with 2 ml BCIP/NBT solution(Sigma, St Louis, Mo.) for 10 minutes. As a positive control, humananti-HCV positive serum was incubated with one strip instead of the Fabpreparations.

None of the tested Fab molecules (from clones 1:5, 1:7, 1:11, L3, L1, A8and A12) reacted to the denatured HCV E2 antigen in the Western blot,indicating that each Fab molecule binds to a conformational epitope ofthe HCV E2 antigen. However, the positive control (human anti-HCVpositive serum) did react with the denatured E2 antigen in the WesternBlot.

The above-described assay was repeated under identical conditions, withthe single change being use of HCV E2 antigen that was gel separatedunder non-denaturing conditions. Both of the clones (1:7 and A8) testedin this further assay were found to bind to the non-denatured E2antigen.

EXAMPLE 11 Inhibition ELISA Assay for Affinity Determination

The affinity of the Fab molecules (from clones 1:5, 1:7, 1:11, L3, L1,A8, and A12) for HCV E2 antigen was estimated using an inhibition ELISAmethod as previously described. Persson et al. (1991) Proc Natl Acad SciUSA 88:2432-2436, Rath et al. (1988) J Immun Methods 106:245-249.Samples to be tested were first titred at ten-fold dilutions in order tobracket a concentration where a ten-fold reduced concentration gave asubstantial reduction in detected binding in the HCV E2 ELISA. Foraffinity measurements, coating of microtitre wells with HCV E2 antigen(HCV genotype 1a) and subsequent blocking was done as described abovefor the ELISA conducted in Example 9. Appropriate dilutions of the Fabsamples, with or without added soluble HCV E2 antigen (finalconcentration 5 μg/ml) were added to the wells, and incubated at ambienttemperature for 3 hours. The plates were washed uniformly 4 times withPBS-T, and developed using AP-anti Fab, substrate, and spectrophotometerreader as described above in Example 9. The reduction of OD in thepresence of soluble HCV E2 antigen was calculated and the concentrationneeded for a 50% reduction estimated by extrapolation.

As depicted in Table III below, the approximate affinities of the Fabmolecules (from clones 1:5, 1:7, 1:11, L3, L1, A8 and A12) for therecombinant conformational HCV E2 antigen (HCV genotype 1a), variedbetween 1×10⁷ and 2×10⁸ M⁻¹.

TABLE III K_(d) ¹ Affinity Clone (nM) (M⁻¹) L3 28 4 × 10⁷ 1:5 >100  <1 ×10⁷  1:7  6 2 × 10⁸  1:11 28 4 × 10⁷ L1 28 4 × 10⁷ A8  6 2 × 10⁸ A12100  1 × 10⁷ ¹Approximate concentration needed of soluble E2 for 50%reduction in OD.

The affinity of the Fab molecules (from clones 1:7, A8 and A12) for adifferent HCV E2 antigen (HCV geneotype 1b) was also assessed using theabove-described inhibition ELISA. The affinities for the E2 antigen ofgenotype 1b in each of the tested molecules was found to be similar tothose reported above (Table III) for the genotype 1a E2 antigen.

In addition, whole recombinant IgG molecules prepared from the followingFab molecule clones: L1; L3; 1:5; 1:7; and 1:11, were assesssed usingthe above-described inhibition ELISA with the HCV genotype 1a E2antigen. The affinities observed were similar to those reported above(Table III) for the Fab molecule clones.

EXAMPLE 12 Inhibition of HCV E2 Binding

The ability of the Fab molecules (from clones 1:5, 1:7, 1:11, L3, L1, A8and A12) to block the binding of HCV E2 to target cells was determinedusing the neutralization of binding (NOB) method of Rosa et al. (1996)Proc Natl Acad Sci USA 93:1759-1763. More particularly, purifiedconformational HCV E2 antigen (from both genotypes HCV 1a and HCV 1b,and prepared as described in Example 3) was used in indirectimmunofluorescence experiments to assess the ability of two separatebatches of bacterially expressed Fab molecule clones to neutralizebinding of the HCV E2 polypeptide to human cells in vitro.

In the assay, 20 μl of the purified conformational HCV E2 antigen (inPBS at 0.5 μg/ml) was mixed with various dilutions of the Fab clones.After incubation at 4° C. for 1 hour, pellets of MOLT-4 cells (a humancell line reported to allow low-level HCV replication in vitro asdescribed by Shimizu et al. (1992) Proc Natl Acad Sci USA 89:5477-5481),were added and the resulting reaction mixture incubated for 1 hour at 4°C. Unnbound HCV antigen and antibodies were removed by twocentrifugations in PBS at 200× g for 5 minutes at 4° C. The cells werethen incubated for 30 minutes at 4° C. with human anti-HCV E2 reactiveserum. The cells were then washed twice in PBS and incubated for 30minutes with fluorescein isothiocyanate-conjugated antiserum specificfor human Fab. The cells were washed again in PBS at 4° C. andresuspended in 100 μl of PBS. Cell-bound fluorescence was analysed witha flow cytometer (FACScan, Becton Dickinson) using Lysis II software(Becton Dickinson). Mean fluorescence intensity of cell populationsincubated with the various Fab preparations were calculated, andcompared to mean fluorescence intensity of cells incubated withoutantibodies or without the E2 antigen.

The results are depicted below in Table IV. As can be seen, all seven ofthe tested Fab clones efficiently inhibited MOLT-4 cell binding by theconformational HCV E2 antigen (both genotypes HCV 1a and HCV 1b). ClonesA8, 1:7, L1 and L3 had very high neutralization activity in the assay.The 50% reduction titer is shown for all tested clones in Table IV, andthe complete assay result for 4 of the clones is shown in FIG. 5. Twonegative control Fab clones, prepared in the same manner as describedabove but directed to HIV-1 envelope glycoprotein gp120 (clones b12 andb14), did not have neutralization activity in the assay. Fab clonesexpressed in eucaryotic cells, and recombinant whole IgG moleculesderived from the Fab clones were found to be negative in a similar NOBassay.

TABLE IV Antigen E2 1a E2 1b clone Exp. I Exp. II Exp. II 1:5 1.5* 2.52.5 1:7 0.02 0.01 0.02 1:11 0.4 0.1 0.15 Ll 0.02 0.03 0.03 L3 0.02 0.020.03 A8 n.d. 0.001 0.007 A12 n.d. 0.1 0.15 b12 >10 >10 >10 b14n.d. >10 >10 *Fab concentration in μg/ml.

Since the first contact between the HCV virus and its host occurs viabinding of the virus envelope to cell-surface receptors, the ability ofthe present Fab molecules to neutralize this interaction establishes theeffectiveness of using those molecules in vaccinations to providepassive immunization to HCV.

Thus, novel human monoclonal antibodies to HCV E2 antigen are disclosed.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

34 132 amino acids amino acid single linear peptide 1 Glu Val Gln LeuLeu Glu Gln Ser Gly Ala Glu Val Arg Lys Pro Gly 1 5 10 15 Ser Ser ValLys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Gly 20 25 30 His Val IleThr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 35 40 45 Met Gly GluSer Ile Pro Ile Phe Gly Ser Ala Asn Tyr Ala Gln Asn 50 55 60 Tyr Ala GlnLys Phe Arg Asp Arg Val Ser Ile Ile Ala Asp Glu Ser 65 70 75 80 Thr SerThr Ser Phe Ile Glu Leu Ser Asn Leu Arg Ser Asp Asp Thr 85 90 95 Ala ValTyr Tyr Cys Ala Arg Asp Pro Pro Arg Tyr Cys Ser Ala Gly 100 105 110 ArgCys Tyr Pro Gly Phe Phe Gln Gln Trp Gly Gln Gly Thr Leu Val 115 120 125Thr Val Ser Ser 130 127 amino acids amino acid single linear peptide 2Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 1015 Ser Ser Val Lys Val Ser Cys Gln Val Phe Gly Asp Thr Phe Ser Arg 20 2530 Tyr Thr Ile Gln Trp Leu Arg Gln Ala Pro Gly Gln Gly Pro Glu Trp 35 4045 Met Gly Asn Ile Ile Pro Val Tyr Asn Thr Pro Asn Tyr Ala Gln Lys 50 5560 Phe Gln Gly Arg Leu Ser Ile Thr Ala Asp Asp Ser Thr Ser Thr Ala 65 7075 80 Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe 8590 95 Cys Ala Arg Val Val Ile Pro Asn Ala Ile Arg His Thr Met Gly Tyr100 105 110 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser115 120 125 128 amino acids amino acid single linear peptide 3 Glu ValGln Leu Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15 SerSer Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Gly 20 25 30 HisVal Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 35 40 45 MetGly Gly Ser Ile Ser Phe Phe Gly Thr Ser Asn Ser Ala Gln Lys 50 55 60 PheGln Gly Arg Val Ser Ile Thr Ala Asp Glu Ser Ala Ser Thr Ala 65 70 75 80Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr 85 90 95Cys Ala Lys Asp Pro Pro Arg Phe Cys Ser Gly Gly Asn Cys Tyr Pro 100 105110 Gly Phe Phe Gln Gln Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115120 125 125 amino acids amino acid single linear peptide 4 Glu Val GlnLeu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Lys Thr Tyr 20 25 30 Gly MetHis Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala GlyIle Ser Phe Asp Gly Ser Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys GlyArg Phe Ile Val Ser Arg Asp Asn Ser Arg Asp Thr Val Phe 65 70 75 80 LeuGln Met Ser Ser Leu Arg Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 AlaThr Glu Gly Ser Pro Phe Gly Ser Ile Lys Gly Arg Tyr Tyr Leu 100 105 110Glu Asn Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 124amino acids amino acid single linear peptide 5 Glu Val Gln Leu Leu GluSer Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Gly Met His Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Trp PheAsp Gly Ser Asn Gln Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe ThrVal Ser Arg Asp Asn Ser Arg Asn Thr Leu Phe 65 70 75 80 Leu Gln Met AsnSer Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu ValLeu Phe Gly Ser Ile Lys Gly Arg Tyr Tyr Leu Glu 100 105 110 Asn Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser 115 120 126 amino acids amino acidsingle linear peptide 6 Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu ValLys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly GlySer Ile Arg Ser Ser 20 25 30 His Trp Trp Ser Trp Val Arg Gln Pro Pro GlyLys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Val Phe Phe Ser Gly Ser Thr IleTyr Asn Pro Ser Leu 50 55 60 Asn Asp Arg Val Phe Met Ser Val Asp Lys SerLys Asp Gln Val Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala AspThr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Ile Lys Met Asn Gln GlyArg Met Met Leu Asp Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr LeuVal Ile Val Ser Ser 115 120 125 126 amino acids amino acid single linearpeptide 7 Glu Val Gln Leu Leu Glu Ser Gly Ser Glu Val Lys Lys Pro GlySer 1 5 10 15 Ser Val Lys Val Ser Cys Arg Ala Ser Gly Gly Ser Phe ArgSer Tyr 20 25 30 Asn Phe Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met 35 40 45 Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Asn Tyr Ala GlnLys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ala ThrGly Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala ValTyr Tyr Cys 85 90 95 Ala Met Pro Tyr Pro Lys His Cys Ser Arg Gly Ser CysTrp Gly Trp 100 105 110 Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser 115 120 125 107 amino acids amino acid single linear peptide 8Ala Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 1 5 1015 Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn Tyr 20 2530 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 4045 Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 5560 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 7075 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Tyr Gly Asn Ser Arg Trp 8590 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 105 aminoacids amino acid single linear peptide 9 Ala Glu Leu Thr Gln Ser Pro AlaThr Leu Ser Leu Ser Pro Gly Glu 1 5 10 15 Arg Ala Thr Leu Ser Cys ArgAla Ser Gln Ser Val Asn Lys Tyr Leu 20 25 30 Ala Trp Tyr Gln Gln Lys ProGly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Ala Ser Asn Arg Ala ThrGly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe ThrLeu Thr Ile Ser Asn Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr TyrCys Gln Gln Arg Ser Asp Trp Val Thr Phe 85 90 95 Gly Gly Gly Thr Lys ValGlu Ile Lys 100 105 107 amino acids amino acid single linear peptide 10Ala Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 1 5 1015 Arg Ala Thr Leu Ser Cys Gly Ala Ser Gln Ser Val Arg Ser Asn Tyr 20 2530 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 4045 Tyr Gly Val Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 5560 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 7075 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg 8590 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 106 aminoacids amino acid single linear peptide 11 Ala Glu Leu Thr Gln Ser ProAla Thr Leu Ser Val Ser Pro Gly Glu 1 5 10 15 Arg Ala Ser Leu Ser CysArg Ala Ser Gln Ser Val Gly Asn Asn Leu 20 25 30 Ala Trp Tyr Gln Gln LysPro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Gly Gly Asn Thr Arg AlaThr Gly Thr Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu PheThr Leu Thr Ile Ser Ser Leu Gln Ser Glu 65 70 75 80 Asp Phe Ala Val TyrPhe Cys Gln His Tyr Ser Thr Trp Pro Leu Thr 85 90 95 Phe Gly Gly Gly ThrLys Val Glu Phe Lys 100 105 107 amino acids amino acid single linearpeptide 12 Ala Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Val GlyGlu 1 5 10 15 Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Ile Tyr SerGly Tyr 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg LeuLeu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg LeuGlu Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly SerPro Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105106 amino acids amino acid single linear peptide 13 Ala Glu Leu Thr GlnSer Pro Ser Ser Leu Ser Ala Phe Val Gly Asp 1 5 10 15 Arg Val Thr IleThr Cys Arg Ala Ser Gln Ser Ile Ser Arg Asn Leu 20 25 30 Asn Trp Tyr GlnGln Lys Pro Gly Thr Ala Pro Lys Val Leu Ile Tyr 35 40 45 Ala Ala Ser SerLeu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly ThrAsp Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro Glu 65 70 75 80 Asp Phe AlaThr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Arg Thr 85 90 95 Phe Gly GlnGly Thr Lys Val Glu Val Lys 100 105 106 amino acids amino acid singlelinear peptide 14 Ala Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu SerPro Gly Glu 1 5 10 15 Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser LeuSer Ser Lys Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala ProArg Leu Phe Ile 35 40 45 Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro AspArg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile SerArg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln TyrGly Thr Pro Arg Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100105 318 base pairs nucleic acid single linear DNA (genomic) 15GAGCTCACGC AGTCTCCAGG CACCCTGTCT TTGTCTCCAG GGGAAAGAGC CACCCTCTCC 60TGCAGGGCCA GTCAGAGTGT TAGCAGCAAT TACTTAGCCT GGTACCAGCA GAGACCTGGC 120CAGGCTCCCA GGCTCCTCAT CTATGGTGCA TCCAGCAGGG CCACTGGCAT CCCAGACAGG 180TTCAGTGGCA GTGGGTCTGG GACAGACTTC ACTCTCACCA TCAGCAGACT GGAGCCTGAA 240GATTTTGCAG TGTATTACTG TCAGCTTTAT GGTAACTCAC GTTGGACGTT CGGCCAAGGG 300ACCAAGGTGG AGATCAAA 318 312 base pairs nucleic acid single linear DNA(genomic) 16 GAGCTCACTC AGTCTCCAGC CACCCTGTCT TTGTCTCCAG GGGAAAGAGCCACCCTCTCC 60 TGCAGGGCCA GTCAGAGTGT TAACAAGTAC TTAGCCTGGT ACCAACAGAAACCTGGCCAG 120 GCTCCCAGGC TCCTCATCTA TGATGCATCC AACAGGGCCA CTGGCATCCCAGCCAGGTTC 180 AGTGGCAGTG GGTCTGGGAC AGACTTCACT CTCACCATCA GCAACCTAGAGCCTGAAGAT 240 TTTGCAGTTT ATTACTGTCA GCAGCGTAGC GACTGGGTCA CTTTCGGCGGAGGGACCAAG 300 GTGGAGATCA AA 312 318 base pairs nucleic acid singlelinear DNA (genomic) 17 GAGCTCACGC AGTCTCCAGG CACCCTGTCT TTGTCTCCAGGGGAAAGAGC CACCCTCTCC 60 TGCGGGGCCA GTCAGAGTGT TAGGAGCAAC TACTTAGCCTGGTACCAGCA AAAACCTGGC 120 CAGGCTCCCA GGCTCCTCAT CTATGGTGTA TCCAGCAGGGCCACTGGCAT CCCAGACAGG 180 TTCAGTGGCA GTGGGTCTGG GACAGACTTC ACTCTCACCATCAGCAGACT GGAGCCTGAA 240 GATTTTGCAG TGTATTACTG TCAGCAGTAT GGTAGCTCACCTCGGACTTT TGGCCAGGGG 300 ACCAAGTTGG AGATCAAA 318 315 base pairs nucleicacid single linear DNA (genomic) 18 GAGCTCACGC AGTCTCCAGC CACCCTGTCTGTGTCTCCAG GGGAAAGAGC CTCCCTCTCC 60 TGCAGGGCCA GTCAGAGTGT CGGTAACAATTTAGCTTGGT ATCAGCAGAA ACCTGGCCAG 120 GCTCCCAGGC TCCTCATTTA TGGTGGAAACACCAGAGCCA CTGGTACCCC AGACAGGTTC 180 AGTGGCAGTG GGTCTGGGAC AGAATTCACTCTCACCATCA GCAGCCTGCA GTCTGAGGAC 240 TTTGCAGTTT ATTTCTGTCA ACACTATAGTACCTGGCCGC TCACTTTCGG CGGGGGGACC 300 AAGGTCGAGT TCAAG 315 372 base pairsnucleic acid single linear DNA (genomic) 19 GAGGTGCAGC TGCTCGAGTCTGGGGGAGGC GTGGTCCAGC CTGGGAGGTC CCTGAGACTC 60 TCCTGTGCAG CGTCTGGATTCACCTTCAGT GCTTATGGCA TGCACTGGGT CCGCCAGGCT 120 CCAGGCAAGG GGCTGGAGTGGGTGGCAGGT ATATGGTTTG ATGGAAGTAA TCAATACTAT 180 TCAGACTCCG TGAAGGGCCGATTCACCGTC TCCAGAGACA ATTCCAGGAA CACGCTGTTT 240 CTGCAAATGA ACAGCCTGAGACCCGAGGAC ACGGCTGTCT ATTACTGTGC GACAGAGGTA 300 CTTTTTGGAT CGATTAAGGGGCGTTACTAC CTTGAAAACT GGGGCCAGGG AACCCTGGTC 360 ACCGTCTCCT CA 372 318base pairs nucleic acid single linear DNA (genomic) 20 GCGGAGCTCACCCAGTCTCC ATCGTCCCTG TCTGCATTTG TNGGAGACAG AGTCACCATC 60 ACTTGCCGGGCAAGTCAGAG TATTAGCAGG AACTTAAATT GGTATCAGCA GAAACCAGGG 120 ACAGCCCCTAAGGTCCTGAT CTATGCTGCA TCCAGTTTGC AAAGTGGGGT CCCATCGAGG 180 TTCAGTGGCAGTGGATCTGG GACAGATTTC ACTCTCACCA TCACCAGTCT GCAACCTGAA 240 GATTTTGCAACTTACTATTG TCAACAGAGT TACACAACCC CTCGGACGTT CGGCCAAGGG 300 ACCAAGGTGGAAGTCAAA 318 318 base pairs nucleic acid single linear DNA (genomic) 21GCCGAGCTCA CGCAGTCTCC AGGCACCCTG TCTTTGTCTC CAGGGGAAAG AGCCACCCTC 60TCCTGCAGGG CCAGTCAGAG TCTTAGCAGC AAATACTTAG CNTGGTACCA ACAGAAACCT 120GGCCAGGCTC CCAGGCTCTT CATTTATGAT GCATCCAGCA GGGCCACTGG CATCCCAGAC 180AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA GCATCAGCAG ATTGGAGCCT 240GAAGATTTTG CAGTGTATTA CTGTCAGCAG TATGGAACAC CTCGCACCTT CGGCCAGGGG 300ACCAAGGTGG AAATCAAA 318 384 base pairs nucleic acid single linear DNA(genomic) 22 CTCGAGCAGT CTGGGGCTGA GGTGAGGAAG CCTGGGTCCT CGGTGAAGGTCTCCTGCAAG 60 GCTTCTGGAG GCACCTTCAG CGGCCATGTT ATCACCTGGG TGCGACAGGCCCCTGGACAA 120 GGACTTGAGT GGATGGGAGA GAGCATCCCT ATCTTTGGTT CCGCAAACTACGCTCAAAAC 180 TACGCTCAGA AATTCCGGGA CAGAGTCTCG ATTATCGCGG ACGAATCCACGAGCACGTCG 240 TTCATTGAGC TGAGCAACCT GAGATCTGAC GACACGGCCG TCTACTACTGTGCGAGAGAC 300 CCTCCAAGAT ATTGCAGTGC TGGTAGATGC TACCCGGGAT TCTTCCAGCAGTGGGGCCAG 360 GGCACCCTCG TCACCGTCTC CTCA 384 369 base pairs nucleicacid single linear DNA (genomic) 23 CTCGAGCAGT CTGGGGCTGA GGTGAAGAAGCCTGGGTCCT CGGTGAAGGT CTCCTGTCAG 60 GTTTTTGGAG ACACCTTCAG CAGATACACTATTCAGTGGT TGCGACAGGC CCCTGGACAA 120 GGGCCTGAGT GGATGGGAAA TATCATCCCTGTCTATAATA CACCAAACTA CGCGCAGAAG 180 TTTCAGGGCA GACTCTCGAT AACCGCCGACGATTCCACGA GCACAGCCTA CATGGAACTG 240 AGTAGCCTCA GATCTGAGGA CACGGCCGTCTATTTCTGTG CGAGAGTCGT AATACCAAAT 300 GCAATCCGGC ACACGATGGG ATATTACTTTGACTACTGGG GCCAGGGAAC CCTGGTCACC 360 GTCTCCTCA 369 372 base pairsnucleic acid single linear DNA (genomic) 24 CTCGAGCAGT CTGGGGCTGAGGTGAAGAAG CCTGGGTCCT CAGTGAAGGT CTCCTGCAAG 60 GCTTCTGGAG GCACCTTCAGCGGCCATGTT ATCAGCTGGG TGCGACAGGC CCCTGGACAA 120 GGGCTTGAGT GGATGGGGGGGAGTATCTCT TTCTTTGGCA CATCAAACTC CGCACAGAAG 180 TTCCAGGGCA GAGTCTCGATTACCGCGGAC GAATCCGCGA GCACAGCCTA CATGGAGCTG 240 AGTAGCCTGA GATCGGAGGACACGGCCATC TATTACTGTG CGAAAGACCC TCCAAGATTT 300 TGTAGTGGTG GTAACTGCTACCCGGGGTTC TTCCAGCAGT GGGGCCAGGG CACCCTGGTC 360 ACCGTCTCCT CA 372 363base pairs nucleic acid single linear DNA (genomic) 25 CTCGAGTCGGGGGGAGGCGT GGTCCAGCCT GGGAGGTCCC TGAGACTCTC CTGTGCAGCG 60 TCTGGATTCACCTTCAAGAC GTATGGCATG CACTGGGTCC GCCAGGCTCC AGGCAAGGGG 120 CTGGAGTGGGTGGCAGGTAT TTCGTTTGAT GGAAGTAACC AATATTACGC AGACTCCGTG 180 AAGGGCCGATTCATCGTCTC CAGAGACAAT TCCAGGGACA CGGTGTTTCT GCAGATGAGC 240 AGCCTGAGACTCGAGGACAC GGCTGTCTAT TACTGTGCGA CAGAGGGTTC TCCTTTTGGC 300 TCGATTAAGGGGCGTTACTA CCTTGAAAAT TGGGGCCAGG GAACCCTGGT CACCGTCTCC 360 TCA 363 378base pairs nucleic acid single linear DNA (genomic) 26 GAGGTGCAGCTGCTCGAGTC GGGCCCAGGA CTGGTGAAGC CTTCGGGGAC CCTGTCCCTC 60 ACCTGCACTGTCTCTGGTGG CTCCATCAGG AGCAGTCACT GGTGGAGTTG GGTCCGCCAG 120 CCCCCAGGGAAGGGACTGGA GTGGATTGGA GAAGTCTTTT TTAGTGGAAG CACCATCTAC 180 AACCCATCCCTCAACGATCG AGTCTTCATG TCTGTAGACA AGTCCAAGGA CCAGGTCTCC 240 CTGAGGCTGAGCTCTGTGAC CGCCGCGGAC ACGGCCGTGT ATTACTGTGC GAGATCCCCC 300 ATAAAAATGAATCAGGGAAG AATGATGTTG GATGCCTTTG ATATCTGGGG CCAGGGGACA 360 CTCGTCATCGTCTCTTCC 378 378 base pairs nucleic acid single linear DNA (genomic) 27GAGGTGCAGC TGCTCGAGTC TGGGTCTGAG GTGAAGAAGC CTGGGTCTTC GGTGAAGGTC 60TCCTGCAGGG CCTCTGGAGG CAGCTTCAGA AGCTACAATT TCAATTGGGT GCGACAGGCC 120CCTGGACAAG GTCTTGAGTG GATGGGAGGC ATCATCCCTA TGTTCGGAAC AGCAAACTAC 180GCACAGAAGT TTCAGGGCAG AGTCACAATT ACCGCGGACG AATCCACGGC CACAGGCTAC 240ATGGAGTTGA GCAGTCTGAG ATCTGAAGAC ACGGCCGTTT ATTACTGTGC GATGCCCTAT 300CCAAAACATT GCAGTCGTGG AAGTTGCTGG GGCTGGTTCG ACCCCTGGGG CCAGGGAACT 360CTGGTCACCG TGTCTTCA 378 47 amino acids amino acid single linear peptide28 Val Ala Gly Val Asp Ala Ser Thr Tyr Thr Thr Gly Gly Gln Ser Gly 1 510 15 Arg Thr Thr Tyr Gly Ile Val Gly Leu Phe Ser Leu Gly Pro Ser Gln 2025 30 Lys Leu Ser Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn Arg 35 4045 22 base pairs nucleic acid single linear DNA (genomic) 29 CAACGCAATTAATGTGAGTT AG 22 24 base pairs nucleic acid single linear DNA (genomic)30 GCCCCCTTAT TAGCGTTTGC CATC 24 21 base pairs nucleic acid singlelinear DNA (genomic) 31 ATAGAAGTTG TTCAGCAGGC A 21 22 base pairs nucleicacid single linear DNA (genomic) 32 AAGACAGCTA TCGCGATTGC AG 22 21 basepairs nucleic acid single linear DNA (genomic) 33 GTCGTTGACC AGGCAGCCCAG 21 21 base pairs nucleic acid single linear DNA (genomic) 34ACCTATTGCC TACGGCAGCC G 21

What is claimed is:
 1. An isolated nucleic acid molecule encoding ahuman Fab molecule, wherein the nucleic acid molecule comprises: a firstnucleotide sequence encoding a first polypeptide that is a bindingportion of a γ1 heavy chain variable region (V_(H)) of said human Fabmolecule where said heavy chain region exhibits immunological bindingaffinity for a hepatitis C Virus (HCV) E2 antigen; and wherein the firstpolypeptide comprises a sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6 and SEQ ID NO:7; and a second nucleotide sequence encoding a secondpolypeptide that is a binding portion of a κ light chain variable region(V_(K)) of said human Fab molecule where said light chain variableregion exhibits immunological binding affinity for a hepatitis C virus(HCV) E2 antigen, and wherein the second polypeptide comprises asequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14,and wherein said Fab molecules have binding affinity greater than 1×10⁷M⁻¹ for HCV E2.
 2. The nucleic acid molecule of claim 1, furthercomprising: a third nucleotide sequence encoding a first leader sequencepeptide, wherein said third nucleotide sequence is operably linked tothe 5′ terminus of the first nucleotide sequence and is capable ofcausing secretion of the encoded heavy chain variable region when theencoded heavy chain variable region and the first leader sequencepeptide are expressed; and a fourth nucleotide sequence encoding asecond leader sequence peptide, wherein said fourth nucleotide sequenceis operably linked to the 5′ terminus of the second nucleotide sequenceand is capable of causing secretion of the encoded light chain variableregion when the encoded light chain variable region and the secondleader sequence peptide are expressed.
 3. The nucleic acid molecule ofclaim 2, wherein the third and fourth nucleotide sequences are selectedfrom the group of leader sequences consisting of omp A and pelB.
 4. Thenucleic acid molecule of claim 1, wherein the first polypeptide sequenceis shown as SEQ ID NO:
 1. 5. The nucleic acid molecule of claim 1,wherein the first polypeptide sequence is shown as SEQ ID NO:
 2. 6. Thenucleic acid molecule of claim 1, wherein the first polypeptide sequenceis shown as SEQ ID NO:
 3. 7. The nucleic acid molecule of claim 1,wherein the first polypeptide sequence is SEQ ID NO:
 4. 8. The nucleicacid molecule of claim 1, wherein the first polypeptide sequence isshown as SEQ ID NO:
 5. 9. The nucleic acid molecule of claim 1, whereinthe first polypeptide sequence is shown as SEQ ID NO:
 6. 10. The nucleicacid molecule f claim 1, wherein the first polypeptide sequence is shownas SEQ ID NO:
 7. 11. The nucleic acid molecule of claim 7, wherein thesecond polypeptide sequence is shown as SEQ ID NO:
 8. 12. The nucleicacid molecule of claim 1, wherein the second polypeptide sequence isshown as SEQ ID NO:
 9. 13. The nucleic acid molecule of claim 1, whereinthe second polypeptide sequence is shown as SEQ ID NO:
 10. 14. Thenucleic acid molecule of claim 1, wherein the second polypeptidesequence is shown as SEQ ID NO:
 11. 15. The nucleic acid molecule ofclaim 1, wherein the second polypeptide sequence is shown as SEQ ID NO:12.
 16. The nucleic acid molecule of claim 1 wherein the secondpolypeptide sequence is shown as SEQ ID NO:
 13. 17. The nucleic acidmolecule of claim 1, wherein the second polypeptide sequence is shown asSEQ ID NO:
 14. 18. An expression vector, comprising the nucleic acidmolecule of claim 1 operably linked to control sequences that direct thetranscription of the first and second nucleotide sequences whereby saidfist and second nucleotide sequences can be transcribed and translatedin a host cell.
 19. The expression vector of claim 18, wherein thecontrol sequences are capable of directing the transcription of thefirst and second nucleotide sequences in a prokaryotic host cell.
 20. Aprokaryotic host cell transformed with the expression vector of claim19.
 21. The expression vector of claim 18, wherein the control sequencesare capable of directing the transcription of the first and secondnucleotide sequences in a eukaryotic host cell.
 22. A eukaryotic hostcell transformed with the expression vector of claim
 21. 23. A method ofproducing a recombinant human Fab molecule, comprising: (a) providing apopulation of transformed host cells according to claim 22; and (b)expressing said recombinant Fab molecule from the expression vector. 24.The isolated nucleic acid molecule of claim 1, wherein the human Fabmolecule encoded by the first and second nucleotide sequences comprisesthe contiguous sequence of amino acids depicted in FIG. 1A (SEQ IDNO: 1) and the contiguous sequence of amino acids depicted in FIG. 2A(SEQ ID NO: 5).
 25. The isolated nucleic acid molecule of claim 1,wherein the human Fab molecule encoded by the first and secondnucleotide sequences comprises the contiguous sequence of amino acidsdepicted in FIG. 1B (SEQ ID NO: 2) and the contiguous sequence of ammoacids depicted in FIG. 2B (SEQ ED NO: 6).
 26. The isolated nucleic acidmolecule of claim 1, wherein the human Fab molecule encoded by the firstand second nucleotide sequences comprises the contiguous sequence ofamino acids depicted in FIG. 1C (SEQ ID NO: 3) and the contiguoussequence of amino acids depicted in FIG. 2C (SEQ ID NO: 7).
 27. Theisolated nucleic acid molecule of claim 1, wherein the human Fabmolecule encoded by the first and second nucleotide sequences comprisesthe contiguous sequence of amino acids depicted in FIG. 1D (SEQ ID NO:4) and the contiguous sequence of amino acids depicted in FIG. 2D (SEQID NO: 8).
 28. A method for providing an antibody titer to HCV in amammalian subject, comprising introducing a therapeutically effectiveamount of the composition comprising the isolated nucleic acid of claim27 to said subject.
 29. An isolated nucleic acid molecule, comprising afirst nucleotide sequence encoding a binding portion of a γ1 heavy chainvariable region (V_(H)) of a human Fab molecule obtained from acombinatorial library, wherein said Fab molecule exhibits immunologicalbinding affinity greater than 1×10⁷ M⁻¹ for a hepatitis C virus (HCV) E2antigen and further wherein the γ1 heavy chain sequence is selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
 30. The nucleic acidmolecule of claim 29, wherein the first polypeptide sequence is shown asSEQ ID NO:
 1. 31. The nucleic acid molecule of claim 29, wherein thefirst polypeptide sequence is shown as SEQ ID NO:
 2. 32. The nucleicacid molecule of claim 29, wherein the first polypeptide sequence shownas SEQ ID NO:
 3. 33. The nucleic acid molecule of claim 29, wherein thefirst polypeptide sequence is shown as SEQ ID NO:
 4. 34. The nucleicacid molecule of claim 29, wherein the first polypeptide sequence isshown as SEQ ID NO:
 5. 35. The nucleic acid molecule of claim 29,wherein the first polypeptide sequence is shown as SEQ ID NO:
 7. 36. Thenucleic acid molecule of claim 29, wherein the first polypeptidesequence is shown as SEQ ID NO:
 7. 37. An expression vector, comprisingthe nucleic acid molecule of claim 29 operably linked to controlsequences that direct the transcription of the first nucleotide sequencewhereby said first nucleotide sequence can be transcribed and translatedin a host cell.
 38. The expression vector of claim 37, wherein thecontrol sequences are capable of directing the transcription of thefirst nucleotide sequence in a prokaryotic host cell.
 39. A prokaryotichost cell transformed with the expression vector of claim
 38. 40. Theexpression vector of claim 37, wherein the control sequences are capableof directing the transcription of the first nucleotide sequence in aeukaryotic host cell.
 41. A eukaryotic host cell transformed with theexpression vector of claim
 40. 42. A method of producing a recombinantpolypeptide having a binding portion of a γ1 heavy chain variable region(V_(H)) of a human Fab molecule, comprising: (a) providing a populationof transformed host cells according to claim 41; and (b) expressing saidrecombinant polypeptide from the expression vector.
 43. An isolatednucleic acid molecule, comprising a first nucleotide sequence encoding abinding portion of a κ light chain variable region (V_(L)) of a humanFab molecule obtained from a combinatorial library, wherein said Fabmolecule exhibits immunological binding affinity greater than 1×10⁷ M⁻¹for a hepatitis C virus (HCV) E2 antigen and further wherein the κ lightchain sequence is selected from the group consisting of SEQ ID NO:8, SEQID NO:9, SEQ ED NO:10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13 and SEQID NO:14.
 44. The nucleic acid molecule of claim 43, wherein the κ lightchain sequence is shown as SEQ ID NO:
 8. 45. The nucleic acid moleculeof claim 43, wherein the κ light chain sequence is shown as SEQ ID NO:9.
 46. The nucleic acid molecule of claim 43, wherein the κ light chainsequence is shown as SEQ ID NO:
 10. 47. The nucleic acid molecule ofclaim 43, wherein the κ light chain sequence is shown as SEQ ID NO: 11.48. The nucleic acid molecule of claim 43, wherein the κ light chainsequence is shown as SEQ ID NO:
 12. 49. The nucleic acid molecule ofclaim 43, wherein the κ light chain sequence is shown as SEQ ID NO: 13.50. The nucleic acid molecule of claim 43, wherein the κ light chainsequence is shown as SEQ ID NO:
 14. 51. An expression vector, comprisingthe nucleic acid molecule of claim 43 operably linked to controlsequences that direct the transcription of the first nucleotide sequencewhereby said first nucleotide sequence can be transcribed and translatedin a host cell.
 52. The expression vector of claim 51, wherein thecontrol sequences are capable of directing the transcription of thefirst nucleotide sequence in a prokaryotic host cell.
 53. A prokaryotichost cell transformed with the expression vector of claim
 52. 54. Theexpression vector of claim 51, wherein the control sequences are capableof directing the transcription of the first nucleotide sequence in aeukaryotic host cell.
 55. A eukaryotic host cell transformed with theexpression vector of claim
 54. 56. A method of producing a recombinantpolypeptide having a binding portion of a κ light chain variable region(V_(L)) of a human Fab molecule, comprising: (a) providing a populationof transformed host cells according to claim 55; and (b) expressing saidrecombinant polypeptide from the expression vector.
 57. An isolatednucleic acid molecule that encodes a recombinant human monoclonalantibody that exhibits immunological binding affinity for a hepatitis Cvirus (HCV) E2 antigen, wherein the antibody comprises at least onegroup of three complementarity determining regions (CDRs) interposedbetween framework regions (FRs) said FRs derived from a humanimmunoglobulin, wherein the group of three CDRs is selected from thegroup consisting of amino acid residue numbers 32-36, 51-71, 104-121 ofSEQ ID NO:1; amino acid residue numbers 32-36, 51-67, 100-116 of SEQ IDNO:2; amino acid residue numbers 32-36, 51-67, 100-117 of SEQ ID NO:3;amino acid residue numbers 31-35, 50-66, 99-114 of SEQ ID NO:4; aminoacid residue numbers 23-34, 49-56, 89-97 of SEQ ID NO:5; amino acidresidue numbers 23-33, 49-55, 88-95 of SEQ ID NO:6; amino acid residuenumbers 23-34, 50-56, 89-97 of SEQ ID NO:7; and amino acid residuenumbers 23-33, 49-55, 88-96 of SEQ ID NO:8.
 58. The isolated nucleicacid molecule of claim 57, wherein the antibody encoded by the nucleicacid molecule comprises a first group of CDRs with amino acid residuenumbers 32-36, 51-71, 104-121 of SEQ ID NO:1 interposed between FRs, anda second group of CDRs with amino acid residue numbers 23-34, 49-56,89-97 of SEQ ID NO:5, interposed between FRs, wherein the first andsecond groups of CDRs interposed between FRs together form a bindingsite for an HCV E2 antigen.
 59. The isolated nucleic acid molecule ofclaim 57, wherein the antibody comprises a first group of CDRs withamino acid residue numbers 32-36, 51-67, 100-116 of SEQ ID NO:2interposed between FRs, and a second group of CDRs with amino acidresidue numbers 23-33, 49-55, 88-95 of SEQ ID NO:6, interposed betweenFRs wherein the first and second groups of CDRs interposed between FRstogether form a binding site for an HCV E2 antigen.
 60. The isolatednucleic acid molecule of claim 57, wherein the antibody comprises afirst group of CDRs with amino acid residue numbers 32-36, 51-67,100-117 of SEQ ID NO:3 interposed between FRs, and a second group ofCDRs with amino acid residue numbers amino acid residue numbers 23-34,50-56, 89-97 of SEQ ID NO:7 interposed between FRs, wherein the firstand second groups of CDRs interposed between FRs together form a bindingsite for an HCV E2 antigen.
 61. The isolated nucleic acid molecule ofclaim 57, wherein the antibody comprises a first group of CDRs withamino acid residue numbers amino acid residue numbers 31-35, 50-66,99-114 of SEQ ID NO:4 interposed between FRs, and a second group of CDRswith amino acid residue numbers 23-33, 49-55, 88-96 of SEQ ID NO:8interposed between FRs, wherein the first and second groups of CDRsinterposed between FRs together form a binding site for an HCV E2antigen.
 62. A method for providing an antibody titer to HCV in amammalian subject, comprising introducing a therapeutically effectiveamount of the vaccine composition of claim 57 to said subject.
 63. Anisolated nucleic acid molecule encoding a human Fab molecule, whereinthe nucleic acid molecule comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. 64.The isolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:15.
 65. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:16.
 66. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:17.
 67. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:18.
 68. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:19.
 69. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:20.
 70. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:21.
 71. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:22.
 72. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:23.
 73. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:24.
 74. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:25.
 75. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:26.
 76. Theisolated nucleic acid molecule of claim 63, wherein the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:27.